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<t>IgD</t> Ligation by Antigen Elicits Basophil Activation (A) <t>ELISA</t> of serum IgD from WT Balb/c (n=29), WT C57BL/6 (n=15), Ighd −/− Balb/c (n=20) or Ighd −/− C57BL/6 (n=10) mice. (B) Left: FCM of CD49b and IgE on basophils (black gate) from circulating CD45 + NTNB cells of a WT Balb/c mouse. NTNB, non-T non-B. Right: FCM of IgD on circulating basophils (blue open profile) from a WT Balb/c mouse. Ctrl, isotype-matched control (gray solid profile). (C) FCM of IgD + basophils from peripheral blood, spleen, bone marrow (BM), lung or mesenteric lymph nodes (MLNs) of WT Balb/c mice (n=5). (D) Imaging FCM of IgD, FcεRI and CD49b from a representative viable Ghost Dye Violet 510 (GV510) − splenic basophil of a WT Balb/c mouse. Scale bar, 5 μm. (E) FCM quantification of FcεRI + CD49b + basophils from BM, blood, spleen or lung CD45 + NTNB cells of WT Balb/c (n=10) or Ighd −/− (n=10) mice. (F) FCM of Il4 -driven GFP expression in splenic or lung FcεRI + CD49b + basophils from WT Balb/c Il4 GFP (n=5) or Il4 GFP Ighd −/− (n=5) mice. MFI, mean fluorescence intensity. (G) ELISA of IL-4 and IL-13 from serum of WT Balb/c or Ighd −/− mice (n=10). (H) Schematics of i.v. reconstitution μMT or Rag2 −/− mice with NP-reactive IgD (NP-IgD) followed by i.p. injection of anti-IgD or i.n. inoculation of NP-OVA. (I) FCM of IgD on splenic FcεRI + CD49b + basophils from a μMT mouse before (ctrl) or after reconstitution as in (H). PBS, control phosphate buffer solution (PBS). (J, K) FCM quantification of total, CD200R3 + or IL-4 + basophils from the spleen of μMT C57BL/6 mice (n=10) (J) or the lungs of IgD-deficient Rag2 −/− (n=10) C57BL/6 mice (K) treated as in (H). (L) FCM quantification of total, IgD + , IgE + or IL-4 + basophils from the lungs of WT Balb/c (n=5) or Ighd −/− (n=5) mice 5 d following i.n. exposure to PBS or recombinant TSLP. (M) ELISA of serum IgD from Balb/c mice following i.v. injection of control empty or TSLP-encoding plasmids (n=10). .
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1) Product Images from "Secreted IgD Amplifies Humoral T Helper-2 Responses by Binding Basophils Via Galectin-9 and CD44"

Article Title: Secreted IgD Amplifies Humoral T Helper-2 Responses by Binding Basophils Via Galectin-9 and CD44

Journal: Immunity

doi: 10.1016/j.immuni.2018.08.013

IgD Ligation by Antigen Elicits Basophil Activation (A) ELISA of serum IgD from WT Balb/c (n=29), WT C57BL/6 (n=15), Ighd −/− Balb/c (n=20) or Ighd −/− C57BL/6 (n=10) mice. (B) Left: FCM of CD49b and IgE on basophils (black gate) from circulating CD45 + NTNB cells of a WT Balb/c mouse. NTNB, non-T non-B. Right: FCM of IgD on circulating basophils (blue open profile) from a WT Balb/c mouse. Ctrl, isotype-matched control (gray solid profile). (C) FCM of IgD + basophils from peripheral blood, spleen, bone marrow (BM), lung or mesenteric lymph nodes (MLNs) of WT Balb/c mice (n=5). (D) Imaging FCM of IgD, FcεRI and CD49b from a representative viable Ghost Dye Violet 510 (GV510) − splenic basophil of a WT Balb/c mouse. Scale bar, 5 μm. (E) FCM quantification of FcεRI + CD49b + basophils from BM, blood, spleen or lung CD45 + NTNB cells of WT Balb/c (n=10) or Ighd −/− (n=10) mice. (F) FCM of Il4 -driven GFP expression in splenic or lung FcεRI + CD49b + basophils from WT Balb/c Il4 GFP (n=5) or Il4 GFP Ighd −/− (n=5) mice. MFI, mean fluorescence intensity. (G) ELISA of IL-4 and IL-13 from serum of WT Balb/c or Ighd −/− mice (n=10). (H) Schematics of i.v. reconstitution μMT or Rag2 −/− mice with NP-reactive IgD (NP-IgD) followed by i.p. injection of anti-IgD or i.n. inoculation of NP-OVA. (I) FCM of IgD on splenic FcεRI + CD49b + basophils from a μMT mouse before (ctrl) or after reconstitution as in (H). PBS, control phosphate buffer solution (PBS). (J, K) FCM quantification of total, CD200R3 + or IL-4 + basophils from the spleen of μMT C57BL/6 mice (n=10) (J) or the lungs of IgD-deficient Rag2 −/− (n=10) C57BL/6 mice (K) treated as in (H). (L) FCM quantification of total, IgD + , IgE + or IL-4 + basophils from the lungs of WT Balb/c (n=5) or Ighd −/− (n=5) mice 5 d following i.n. exposure to PBS or recombinant TSLP. (M) ELISA of serum IgD from Balb/c mice following i.v. injection of control empty or TSLP-encoding plasmids (n=10). .
Figure Legend Snippet: IgD Ligation by Antigen Elicits Basophil Activation (A) ELISA of serum IgD from WT Balb/c (n=29), WT C57BL/6 (n=15), Ighd −/− Balb/c (n=20) or Ighd −/− C57BL/6 (n=10) mice. (B) Left: FCM of CD49b and IgE on basophils (black gate) from circulating CD45 + NTNB cells of a WT Balb/c mouse. NTNB, non-T non-B. Right: FCM of IgD on circulating basophils (blue open profile) from a WT Balb/c mouse. Ctrl, isotype-matched control (gray solid profile). (C) FCM of IgD + basophils from peripheral blood, spleen, bone marrow (BM), lung or mesenteric lymph nodes (MLNs) of WT Balb/c mice (n=5). (D) Imaging FCM of IgD, FcεRI and CD49b from a representative viable Ghost Dye Violet 510 (GV510) − splenic basophil of a WT Balb/c mouse. Scale bar, 5 μm. (E) FCM quantification of FcεRI + CD49b + basophils from BM, blood, spleen or lung CD45 + NTNB cells of WT Balb/c (n=10) or Ighd −/− (n=10) mice. (F) FCM of Il4 -driven GFP expression in splenic or lung FcεRI + CD49b + basophils from WT Balb/c Il4 GFP (n=5) or Il4 GFP Ighd −/− (n=5) mice. MFI, mean fluorescence intensity. (G) ELISA of IL-4 and IL-13 from serum of WT Balb/c or Ighd −/− mice (n=10). (H) Schematics of i.v. reconstitution μMT or Rag2 −/− mice with NP-reactive IgD (NP-IgD) followed by i.p. injection of anti-IgD or i.n. inoculation of NP-OVA. (I) FCM of IgD on splenic FcεRI + CD49b + basophils from a μMT mouse before (ctrl) or after reconstitution as in (H). PBS, control phosphate buffer solution (PBS). (J, K) FCM quantification of total, CD200R3 + or IL-4 + basophils from the spleen of μMT C57BL/6 mice (n=10) (J) or the lungs of IgD-deficient Rag2 −/− (n=10) C57BL/6 mice (K) treated as in (H). (L) FCM quantification of total, IgD + , IgE + or IL-4 + basophils from the lungs of WT Balb/c (n=5) or Ighd −/− (n=5) mice 5 d following i.n. exposure to PBS or recombinant TSLP. (M) ELISA of serum IgD from Balb/c mice following i.v. injection of control empty or TSLP-encoding plasmids (n=10). .

Techniques Used: Ligation, Activation Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay, Imaging, Expressing, Fluorescence, Injection, Recombinant

Basophil-Bound IgD Interacts with Galectin-9 and Its Ligation Induces Th2 Cell-Associated Cytokine Expression But Inhibits Cytoskeleton Remodeling (A) Schematics of i.p. anti-IgD treated mice i.v. reconstituted or not with secreted IgD. (B-C) ELISA of serum IgG1, IgE (B), IL-4 and IL-13 (C) from WT Balb/c (n=10) or Ighd −/− (n=10) mice in the presence or absence of i.v. reconstitution with secreted IgD (NP-IgD), followed by i.p. injection of anti-IgD. Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to onset of anti-IgD treatment (day 0). (D) IFA of human tonsillar tissue stained for IgD (green), IgM (red), cytokeratin (purple) and nuclei (blue). EP, epithelium; FO, follicle; PC, plasma cell. Original magnification, ×20 (top) or ×63 (bottom). Scale bars, 50 μm (top) and 5 μm (bottom). (E) ELISA of IgD specific to α-s-casein, β-lactoglobulin (β-LGB) or α-lactalbumin (α-LAL) from plasma of FPIES children with (n=5) or without (n=5) dietary milk restrictions. (F) FCM of IgD (red open profile) bound to human tonsillar or circulating CD123 + FcεRI + basophils (red gate). Gray open profiles, control isotype-matched antibody with irrelevant binding activity. (G) Microarray analysis of genes expressed by human basophils upon IgD cross-linking. The Volcano plot represents genes differentially expressed by basophils treated with anti-IgD or a F(ab’)2 control antibody (ctrl). Red and blue dots, up-regulated and down-regulated genes, respectively; FC, fold change. (H) Heat map of coordinated gene sets identified by gene set enrichment analysis in human basophils treated as in (G). NES (normalized enrichment score) indicates correlation between individual gene sets. Positive correlation, NES > 0 (yellow gradient); negative correlation, NES
Figure Legend Snippet: Basophil-Bound IgD Interacts with Galectin-9 and Its Ligation Induces Th2 Cell-Associated Cytokine Expression But Inhibits Cytoskeleton Remodeling (A) Schematics of i.p. anti-IgD treated mice i.v. reconstituted or not with secreted IgD. (B-C) ELISA of serum IgG1, IgE (B), IL-4 and IL-13 (C) from WT Balb/c (n=10) or Ighd −/− (n=10) mice in the presence or absence of i.v. reconstitution with secreted IgD (NP-IgD), followed by i.p. injection of anti-IgD. Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to onset of anti-IgD treatment (day 0). (D) IFA of human tonsillar tissue stained for IgD (green), IgM (red), cytokeratin (purple) and nuclei (blue). EP, epithelium; FO, follicle; PC, plasma cell. Original magnification, ×20 (top) or ×63 (bottom). Scale bars, 50 μm (top) and 5 μm (bottom). (E) ELISA of IgD specific to α-s-casein, β-lactoglobulin (β-LGB) or α-lactalbumin (α-LAL) from plasma of FPIES children with (n=5) or without (n=5) dietary milk restrictions. (F) FCM of IgD (red open profile) bound to human tonsillar or circulating CD123 + FcεRI + basophils (red gate). Gray open profiles, control isotype-matched antibody with irrelevant binding activity. (G) Microarray analysis of genes expressed by human basophils upon IgD cross-linking. The Volcano plot represents genes differentially expressed by basophils treated with anti-IgD or a F(ab’)2 control antibody (ctrl). Red and blue dots, up-regulated and down-regulated genes, respectively; FC, fold change. (H) Heat map of coordinated gene sets identified by gene set enrichment analysis in human basophils treated as in (G). NES (normalized enrichment score) indicates correlation between individual gene sets. Positive correlation, NES > 0 (yellow gradient); negative correlation, NES

Techniques Used: Ligation, Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection, Concentration Assay, Immunofluorescence, Staining, Binding Assay, Activity Assay, Microarray

IgD Binds to Basophils through Galectin-9 and CD44 (A) IB of galectin-9 following IP of human IgD and galectin-9 protein mix with control (ctrl) or anti-IgD antibodies. Prior to IP, the protein mix was supplemented with control PBS, glucose or lactose. (B) FCM of human IgD on human KU812 cells cultured for 30 min with control medium alone (ctrl), IgD or an IgD-galectin-9 complex formed by pre-incubating IgD with galectin-9 for 10 min. (C) FCM of human IgD or galectin-9 on KU812 cells cultured with IgD-galectin-9 in the presence of medium alone (ctrl), glucose or lactose for 30 min. (D) FCM of human IgD or CD44 on KU812 cells treated with scrambled (ctrl) or CD44-targeting small interfering RNA (siRNA) and later incubated with or without (ctrl) IgD-galectin-9. MFI, mean fluorescence intensity. (E) Confocal imaging of human basophils stained for IgD (blue), galectin-9 (green) and CD44 (red). Scale bar, 0.5 μm. (F) FCM of IgD on human basophils incubated with or without (ctrl) IgD-galectin-9 for 30 min. (G) ELISA of IL-4 from human basophils incubated with medium alone (ctrl) and with or without the IgD-galectin-9 complex in the presence or absence of IgD cross-linking by anti-IgD for 18 h. (H, I) FCM of IgD + basophils from the spleen or lung of WT C57BL/6, Lgals9 −/− (H) or Cd44 −/− (I) mice. (J, K) ELISA of serum IgG1 and IgE to NP from WT Balb/c (n=10), Lgals9 −/− (n=8) (J) or Cd44 −/− (n=10) (K) mice following s.c. immunization with NP-OVA and papain. Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to the onset of anti-IgD treatment (day 0). (L) ELISA of serum total IgG1 and IgE from WT C57BL/6 controls (n=10) and Lgals9 −/− mice (n=8) following i.p. injection of anti-IgD. .
Figure Legend Snippet: IgD Binds to Basophils through Galectin-9 and CD44 (A) IB of galectin-9 following IP of human IgD and galectin-9 protein mix with control (ctrl) or anti-IgD antibodies. Prior to IP, the protein mix was supplemented with control PBS, glucose or lactose. (B) FCM of human IgD on human KU812 cells cultured for 30 min with control medium alone (ctrl), IgD or an IgD-galectin-9 complex formed by pre-incubating IgD with galectin-9 for 10 min. (C) FCM of human IgD or galectin-9 on KU812 cells cultured with IgD-galectin-9 in the presence of medium alone (ctrl), glucose or lactose for 30 min. (D) FCM of human IgD or CD44 on KU812 cells treated with scrambled (ctrl) or CD44-targeting small interfering RNA (siRNA) and later incubated with or without (ctrl) IgD-galectin-9. MFI, mean fluorescence intensity. (E) Confocal imaging of human basophils stained for IgD (blue), galectin-9 (green) and CD44 (red). Scale bar, 0.5 μm. (F) FCM of IgD on human basophils incubated with or without (ctrl) IgD-galectin-9 for 30 min. (G) ELISA of IL-4 from human basophils incubated with medium alone (ctrl) and with or without the IgD-galectin-9 complex in the presence or absence of IgD cross-linking by anti-IgD for 18 h. (H, I) FCM of IgD + basophils from the spleen or lung of WT C57BL/6, Lgals9 −/− (H) or Cd44 −/− (I) mice. (J, K) ELISA of serum IgG1 and IgE to NP from WT Balb/c (n=10), Lgals9 −/− (n=8) (J) or Cd44 −/− (n=10) (K) mice following s.c. immunization with NP-OVA and papain. Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to the onset of anti-IgD treatment (day 0). (L) ELISA of serum total IgG1 and IgE from WT C57BL/6 controls (n=10) and Lgals9 −/− mice (n=8) following i.p. injection of anti-IgD. .

Techniques Used: Cell Culture, Small Interfering RNA, Incubation, Fluorescence, Imaging, Staining, Enzyme-linked Immunosorbent Assay, Mouse Assay, Concentration Assay, Injection

Ligation of Basophil-Bound IgD by Antigen Enhances IgG1 and IgE Responses (A) Schematics of i.p. immunization with NP-OVA and papain. (B) ELISA of serum OVA-specific IgG1 and IgE from WT Balb/c (n=10) or Ighd −/− (n=10) mice immunized as in (A). Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to the onset of immunization (day 0). (C-D) FCM quantification of total and IL-4-expressing splenic PD-1 high CXCR5 high Tfh cells from WT Balb/c (n=5) or Ighd −/− (n=5) mice immunized as in (A). (E, F) ELISA of serum NP-specific IgG1 from WT Balb/c (n=10) or Ighd −/− (n=10) mice immunized as in (A). BSA haptenated with 4 or 16 NPs was used to measure high-affinity (HA) and both HA and lowaffinity (LA) IgG1, respectively. (G) IFA of splenic tissue from immunized Balb/c mice stained for IgD (green), IgM (red) and nuclei (blue) following i.p. immunization as in (B). Inset: IgD + IgM − plasmablast next to IgD − IgM + plasmablast. FO, follicle. Original magnification, ×10 with ×2 enlargement. Scale bar, 50 μm. (H) ELISPOT of spleen ASCs expressing NP-specific IgD from WT Balb/c (n=5) or Ighd −/− (n=5) mice 3 d following i.p. immunization with PBS or NP-OVA and papain. (I) Schematics of i.v. reconstitution with NP-reactive IgD (NP-IgD) followed by i.p. immunization with NP-OVA and papain. (J) ELISA of serum OVA-specific IgG1 and IgE from WT Balb/c controls (n=10), Ighd −/− mice (n=10) or NP-IgD-reconstituted Ighd −/− mice (n=10) following i.p. immunization with NP-OVA and papain as in (I). (K) FCM of circulating FcεRI + IgE + basophils from WT Balb/c mice after i.v. injection of a control (ctrl) IgG2b antibody or a basophil-depleting anti-CD200R3 antibody. (L) ELISA of serum total IgD as well as serum NP-specific IgG1 and IgE in control (n=10) or basophil-depleted (n=10) WT Balb/c mice after i.p. immunization with NP-OVA and papain. .
Figure Legend Snippet: Ligation of Basophil-Bound IgD by Antigen Enhances IgG1 and IgE Responses (A) Schematics of i.p. immunization with NP-OVA and papain. (B) ELISA of serum OVA-specific IgG1 and IgE from WT Balb/c (n=10) or Ighd −/− (n=10) mice immunized as in (A). Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to the onset of immunization (day 0). (C-D) FCM quantification of total and IL-4-expressing splenic PD-1 high CXCR5 high Tfh cells from WT Balb/c (n=5) or Ighd −/− (n=5) mice immunized as in (A). (E, F) ELISA of serum NP-specific IgG1 from WT Balb/c (n=10) or Ighd −/− (n=10) mice immunized as in (A). BSA haptenated with 4 or 16 NPs was used to measure high-affinity (HA) and both HA and lowaffinity (LA) IgG1, respectively. (G) IFA of splenic tissue from immunized Balb/c mice stained for IgD (green), IgM (red) and nuclei (blue) following i.p. immunization as in (B). Inset: IgD + IgM − plasmablast next to IgD − IgM + plasmablast. FO, follicle. Original magnification, ×10 with ×2 enlargement. Scale bar, 50 μm. (H) ELISPOT of spleen ASCs expressing NP-specific IgD from WT Balb/c (n=5) or Ighd −/− (n=5) mice 3 d following i.p. immunization with PBS or NP-OVA and papain. (I) Schematics of i.v. reconstitution with NP-reactive IgD (NP-IgD) followed by i.p. immunization with NP-OVA and papain. (J) ELISA of serum OVA-specific IgG1 and IgE from WT Balb/c controls (n=10), Ighd −/− mice (n=10) or NP-IgD-reconstituted Ighd −/− mice (n=10) following i.p. immunization with NP-OVA and papain as in (I). (K) FCM of circulating FcεRI + IgE + basophils from WT Balb/c mice after i.v. injection of a control (ctrl) IgG2b antibody or a basophil-depleting anti-CD200R3 antibody. (L) ELISA of serum total IgD as well as serum NP-specific IgG1 and IgE in control (n=10) or basophil-depleted (n=10) WT Balb/c mice after i.p. immunization with NP-OVA and papain. .

Techniques Used: Ligation, Enzyme-linked Immunosorbent Assay, Mouse Assay, Concentration Assay, Expressing, Immunofluorescence, Staining, Enzyme-linked Immunospot, Injection

IgD Ligation by Antigen Induces Basophil Expression of IL-4 (A) Schematics of s.c. immunization with NP-OVA combined with control PBS, papain or NP-IgD. (B) Confocal imaging of CD169 (subcapsular sinus macrophage molecule, red), B220 (B cell molecule, blue) and Mcpt8 (YFP, yellow) from draining lymph node (DLN) of a C57BL/6 Mcpt8 YFP mouse immunized for 6 h as in (A). FO, follicle; dashed line, follicular border. Original magnification, ×5; rightbottom panel, ×40. Scale bars, 50 μm (top and bottom-left panels) or 5 μm (bottom-right panel). (C-E) FCM quantification of total YFP + or GFP + basophils and qRT-PCR quantification of Il4 transcripts encoding IL-4 from the DLN of C57BL/6 Mcpt8 YFP (n=5) mice (C, D) or Balb/c Il4 GFP (n=10) mice (E) 6 h following s.c. immunization as in (A). qRT-PCR results (D, bottom graph) are presented as relative expression (RE) compared to mRNA for glyceraldeheyde phosphate dehydrogenase (GAPDH). (F) Schematics of s.c. immunization with NP-OVA combined with papain, alum or CFA. (G) ELISA of serum NP-specific IgD from WT Balb/c mice (n=17) following s.c. immunization with NP-OVA and papain. PI, pre-immune (day −1 relatively to the onset of immunization (day 0). (H) ELISA of serum NP-specific IgG1 and IgE from WT Balb/c (n=12) or Ighd −/− (n=16) mice following s.c. immunization with NP-OVA and papain. (I) ELISA of serum NP-specific IgG1 and IgE from WT Balb/c (n=15) or Ighd −/− (n=15) mice following s.c. immunization with NP-OVA and alum. (J) ELISA of serum NP-specific IgG2a and IgG2b from Balb/c (n=10) or Ighd −/− (n=10) mice following s.c. immunization with NP-OVA and CFA. Data show one experiment of three with similar results (B, C) or summarize results from two experiments with 5–10 (D, E) or 10–17 (G-J) mice per experimental group. Results are presented as mean ± SEM; *p
Figure Legend Snippet: IgD Ligation by Antigen Induces Basophil Expression of IL-4 (A) Schematics of s.c. immunization with NP-OVA combined with control PBS, papain or NP-IgD. (B) Confocal imaging of CD169 (subcapsular sinus macrophage molecule, red), B220 (B cell molecule, blue) and Mcpt8 (YFP, yellow) from draining lymph node (DLN) of a C57BL/6 Mcpt8 YFP mouse immunized for 6 h as in (A). FO, follicle; dashed line, follicular border. Original magnification, ×5; rightbottom panel, ×40. Scale bars, 50 μm (top and bottom-left panels) or 5 μm (bottom-right panel). (C-E) FCM quantification of total YFP + or GFP + basophils and qRT-PCR quantification of Il4 transcripts encoding IL-4 from the DLN of C57BL/6 Mcpt8 YFP (n=5) mice (C, D) or Balb/c Il4 GFP (n=10) mice (E) 6 h following s.c. immunization as in (A). qRT-PCR results (D, bottom graph) are presented as relative expression (RE) compared to mRNA for glyceraldeheyde phosphate dehydrogenase (GAPDH). (F) Schematics of s.c. immunization with NP-OVA combined with papain, alum or CFA. (G) ELISA of serum NP-specific IgD from WT Balb/c mice (n=17) following s.c. immunization with NP-OVA and papain. PI, pre-immune (day −1 relatively to the onset of immunization (day 0). (H) ELISA of serum NP-specific IgG1 and IgE from WT Balb/c (n=12) or Ighd −/− (n=16) mice following s.c. immunization with NP-OVA and papain. (I) ELISA of serum NP-specific IgG1 and IgE from WT Balb/c (n=15) or Ighd −/− (n=15) mice following s.c. immunization with NP-OVA and alum. (J) ELISA of serum NP-specific IgG2a and IgG2b from Balb/c (n=10) or Ighd −/− (n=10) mice following s.c. immunization with NP-OVA and CFA. Data show one experiment of three with similar results (B, C) or summarize results from two experiments with 5–10 (D, E) or 10–17 (G-J) mice per experimental group. Results are presented as mean ± SEM; *p

Techniques Used: Ligation, Expressing, Imaging, Quantitative RT-PCR, Mouse Assay, Enzyme-linked Immunosorbent Assay

IgD Attenuates Acute Lung Inflammation Following Secondary Antigen Exposure (A) Schematics of i.p. OVA sensitization followed by a challenge consisting of four consecutive i.t. inoculations of OVA or control PBS. (B) ELISA of OVA-specific IgD and IgE from serum of WT Balb/c (n=10) or Ighd −/− (n=10) mice treated as in (A). Dashed line, maximum antibody concentration 4 hs after the last i.t. inoculation of PBS. (C) FCM quantitation of IgE on l basophils from lungs of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). (D) Microscopic quantitation of total cells from the bronchoalveolar lavage (BAL) of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). (E, F) FCM quantitation of CD49b + IgE + basophils from lungs of WT Balb/c (n=10) or Ighd −/− (n=10) mice treated as in (A). NTNB, non-T non-B. (G) ELISA of Mcpt8 from serum of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). (H, I) FCM quantitation of CD45 + Siglec-F + eosinophils from lungs of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). Data summarize two experiments with at least five mice per experimental group. Results are presented as mean ± SEM; *p
Figure Legend Snippet: IgD Attenuates Acute Lung Inflammation Following Secondary Antigen Exposure (A) Schematics of i.p. OVA sensitization followed by a challenge consisting of four consecutive i.t. inoculations of OVA or control PBS. (B) ELISA of OVA-specific IgD and IgE from serum of WT Balb/c (n=10) or Ighd −/− (n=10) mice treated as in (A). Dashed line, maximum antibody concentration 4 hs after the last i.t. inoculation of PBS. (C) FCM quantitation of IgE on l basophils from lungs of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). (D) Microscopic quantitation of total cells from the bronchoalveolar lavage (BAL) of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). (E, F) FCM quantitation of CD49b + IgE + basophils from lungs of WT Balb/c (n=10) or Ighd −/− (n=10) mice treated as in (A). NTNB, non-T non-B. (G) ELISA of Mcpt8 from serum of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). (H, I) FCM quantitation of CD45 + Siglec-F + eosinophils from lungs of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). Data summarize two experiments with at least five mice per experimental group. Results are presented as mean ± SEM; *p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Concentration Assay, Quantitation Assay

2) Product Images from "Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response"

Article Title: Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response

Journal: Pathogens

doi: 10.3390/pathogens6020016

In vitro immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on the Ig secretion by naive B cells induced by CpG ODNs. Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) CpG ODNs (1 µg/mL). After seven days of incubation, supernatants were collected and total IgM ( A ) and IgG ( B ) were quantified by ELISA. Cells stimulated with CpG ODNs alone (black bars) served as the positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of stimulation by CpG ODNs. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panel) experiments.
Figure Legend Snippet: In vitro immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on the Ig secretion by naive B cells induced by CpG ODNs. Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) CpG ODNs (1 µg/mL). After seven days of incubation, supernatants were collected and total IgM ( A ) and IgG ( B ) were quantified by ELISA. Cells stimulated with CpG ODNs alone (black bars) served as the positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of stimulation by CpG ODNs. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panel) experiments.

Techniques Used: In Vitro, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Positive Control

Titration of capsular polysaccharide (CPS)-specific antibodies in mice after infection with S. suis serotype 2, S. suis serotype 14, group B Streptococcus (GBS) serotype III or GBS serotype V. Mice were infected with 2 × 10 7 CFU of live S. suis serotype 2 strain P1/7 ( n = 60) ( A ); 5 × 10 6 CFU of live S. suis serotype 14 strain DAN13730 ( n = 40) ( B ); 2 × 10 6 CFU of live GBS serotype III strain COH-1 ( n = 40) ( C ); or 10 4 CFU of live GBS serotype V strain CJB111 ( n = 40) ( D ). Total Ig (IgG plus IgM) anti-CPS titers were determined by ELISA on Days 7 (in blue), 14 (in green) and 21 (in red) in surviving mice. IgM and IgG anti-CPS titers were determined on Day 21 (in red). For comparative purpose, total Ig (IgG plus IgM), IgM and IgG anti-protein (‘prot’) titers were also determined on Day 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. “C − ” represents a pool of control mice ( n = 3) injected with vehicle solution, whose titers were evaluated on Days 7, 14 and 21. * Statistically significant difference ( p
Figure Legend Snippet: Titration of capsular polysaccharide (CPS)-specific antibodies in mice after infection with S. suis serotype 2, S. suis serotype 14, group B Streptococcus (GBS) serotype III or GBS serotype V. Mice were infected with 2 × 10 7 CFU of live S. suis serotype 2 strain P1/7 ( n = 60) ( A ); 5 × 10 6 CFU of live S. suis serotype 14 strain DAN13730 ( n = 40) ( B ); 2 × 10 6 CFU of live GBS serotype III strain COH-1 ( n = 40) ( C ); or 10 4 CFU of live GBS serotype V strain CJB111 ( n = 40) ( D ). Total Ig (IgG plus IgM) anti-CPS titers were determined by ELISA on Days 7 (in blue), 14 (in green) and 21 (in red) in surviving mice. IgM and IgG anti-CPS titers were determined on Day 21 (in red). For comparative purpose, total Ig (IgG plus IgM), IgM and IgG anti-protein (‘prot’) titers were also determined on Day 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. “C − ” represents a pool of control mice ( n = 3) injected with vehicle solution, whose titers were evaluated on Days 7, 14 and 21. * Statistically significant difference ( p

Techniques Used: Titration, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Injection

Titration of CPS-specific antibodies in mice after immunization with purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS. Mice ( n = 8) were immunized with 2 µg of purified native or desialylated S. suis serotype 2 ( A ); S. suis serotype 14 ( B ); GBS serotype III ( C ); or GBS serotype V ( D ) CPS emulsified with STIMUNE ® . Total Ig (IgG plus IgM) anti-native CPS titers were determined by ELISA on Days 7, 14 and 21. “C − ” represents a pool of control mice ( n = 3) injected with STIMUNE ® only, whose titers were evaluated on Days 7, 14 and 21. Data from individual mice are presented, including the geometric mean with 95% confidence interval. * Statistically significant difference ( p
Figure Legend Snippet: Titration of CPS-specific antibodies in mice after immunization with purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS. Mice ( n = 8) were immunized with 2 µg of purified native or desialylated S. suis serotype 2 ( A ); S. suis serotype 14 ( B ); GBS serotype III ( C ); or GBS serotype V ( D ) CPS emulsified with STIMUNE ® . Total Ig (IgG plus IgM) anti-native CPS titers were determined by ELISA on Days 7, 14 and 21. “C − ” represents a pool of control mice ( n = 3) injected with STIMUNE ® only, whose titers were evaluated on Days 7, 14 and 21. Data from individual mice are presented, including the geometric mean with 95% confidence interval. * Statistically significant difference ( p

Techniques Used: Titration, Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay, Injection

Adjuvant effect of CpG oligodeoxynucleotides (ODNs) on the CPS-specific humoral response in mice immunized with purified native or desialylated S. suis serotype 2 CPS or purified S. pneumoniae serotype 3 CPS (PS3). ( A ) In a first set of experiments, mice ( n = 8) were immunized with 2 µg of purified native (n) or desialylated (dS) S. suis serotype 2 CPS (CPS S. suis ) or PS3 in PBS on Day 0 and 80 µg of CpG ODNs two days after. The control group ( n = 8) received CPS or PS3 on Day 0 and PBS two days later. The placebo group ( n = 3) received PBS or CpG ODNs only. Total Ig (IgG plus IgM) anti-native S. suis type 2 CPS or anti-PS3 titers were determined by ELISA on Days 7, 14 and 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. ( B ) In a second set of experiments, mice ( n = 5) were immunized as described in (A), but splenocytes were collected on Day 5 after immunization, and anti-CPS antibody-secreting cells (ASCs) were enumerated by ELISpot as described in the Materials and Methods section. Data are expressed as arithmetic means with SEM. ( C ) Visualization of PS3 (top) or native S. suis type 2 CPS (bottom) specific ASCs in ELISpot wells from splenocytes of mice obtained in (B). * p
Figure Legend Snippet: Adjuvant effect of CpG oligodeoxynucleotides (ODNs) on the CPS-specific humoral response in mice immunized with purified native or desialylated S. suis serotype 2 CPS or purified S. pneumoniae serotype 3 CPS (PS3). ( A ) In a first set of experiments, mice ( n = 8) were immunized with 2 µg of purified native (n) or desialylated (dS) S. suis serotype 2 CPS (CPS S. suis ) or PS3 in PBS on Day 0 and 80 µg of CpG ODNs two days after. The control group ( n = 8) received CPS or PS3 on Day 0 and PBS two days later. The placebo group ( n = 3) received PBS or CpG ODNs only. Total Ig (IgG plus IgM) anti-native S. suis type 2 CPS or anti-PS3 titers were determined by ELISA on Days 7, 14 and 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. ( B ) In a second set of experiments, mice ( n = 5) were immunized as described in (A), but splenocytes were collected on Day 5 after immunization, and anti-CPS antibody-secreting cells (ASCs) were enumerated by ELISpot as described in the Materials and Methods section. Data are expressed as arithmetic means with SEM. ( C ) Visualization of PS3 (top) or native S. suis type 2 CPS (bottom) specific ASCs in ELISpot wells from splenocytes of mice obtained in (B). * p

Techniques Used: Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

In vivo immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on ovalbumin (OVA)-specific antibody response. Mice ( n = 8) were co-immunized intraperitoneally with 10 µg of OVA and 2 µg of purified native S. suis serotype 2 ( A ), S. suis serotype 14 ( B ), GBS serotype III ( C ) or GBS serotype V ( D ) CPS in PBS on Days 0 and 21. Control group ( n = 8) received OVA only on Days 0 and 21. Total Ig (IgG plus IgM) anti-OVA titers were determined by ELISA on Days 7, 14, 21 and 35. “C − ” represents a pool of placebo mice ( n = 3) injected with PBS, whose titers were evaluated on Days 7, 14, 21 and 35. Data from individual mice are presented, including the geometric mean with 95% confidence interval. An arrow indicates secondary immunization. * p
Figure Legend Snippet: In vivo immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on ovalbumin (OVA)-specific antibody response. Mice ( n = 8) were co-immunized intraperitoneally with 10 µg of OVA and 2 µg of purified native S. suis serotype 2 ( A ), S. suis serotype 14 ( B ), GBS serotype III ( C ) or GBS serotype V ( D ) CPS in PBS on Days 0 and 21. Control group ( n = 8) received OVA only on Days 0 and 21. Total Ig (IgG plus IgM) anti-OVA titers were determined by ELISA on Days 7, 14, 21 and 35. “C − ” represents a pool of placebo mice ( n = 3) injected with PBS, whose titers were evaluated on Days 7, 14, 21 and 35. Data from individual mice are presented, including the geometric mean with 95% confidence interval. An arrow indicates secondary immunization. * p

Techniques Used: In Vivo, Purification, Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection

In vitro immunomodulatory effect of purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on BAFF/IL-4-induced Ig secretion by naive B cells. ( A , B ) Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) BAFF (1 µg/mL) along with IL-4 (50 ng/mL); ( C , D ) in order to evaluate the influence of sialic acid, cells were also incubated with desialylated (dS) S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS in parallel to cells incubated with the respective native CPS (n) as described in ( A , B ). After seven days of incubation, supernatants were collected, and total IgM ( A , C ) and IgG ( B , D ) was quantified by ELISA. Cells stimulated with BAFF/IL-4 alone (black bars) served as a positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of BAFF/IL-4 stimulation. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panels) experiments. * p
Figure Legend Snippet: In vitro immunomodulatory effect of purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on BAFF/IL-4-induced Ig secretion by naive B cells. ( A , B ) Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) BAFF (1 µg/mL) along with IL-4 (50 ng/mL); ( C , D ) in order to evaluate the influence of sialic acid, cells were also incubated with desialylated (dS) S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS in parallel to cells incubated with the respective native CPS (n) as described in ( A , B ). After seven days of incubation, supernatants were collected, and total IgM ( A , C ) and IgG ( B , D ) was quantified by ELISA. Cells stimulated with BAFF/IL-4 alone (black bars) served as a positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of BAFF/IL-4 stimulation. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panels) experiments. * p

Techniques Used: In Vitro, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Positive Control

3) Product Images from "CD180 Ligation Inhibits TLR7- and TLR9-Mediated Activation of Macrophages and Dendritic Cells Through the Lyn-SHP-1/2 Axis in Murine Lupus"

Article Title: CD180 Ligation Inhibits TLR7- and TLR9-Mediated Activation of Macrophages and Dendritic Cells Through the Lyn-SHP-1/2 Axis in Murine Lupus

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02643

Treatment effect of anti-CD180 Ab on lupus-symptoms of MRL/ lpr mice. (A) Proteinuria was collected weekly and determined by ELISA. (B) Representative images of marked splenomegaly in all groups of mice. (C,D) ELISA analysis of the anti-dsDNA antibody (C) and anti-RNA antibody (D) in serum. (E) H E staining of the kidney sections from all groups of mice. (F) Immunofluorescence staining to detect IgG deposit in the kidney sections. (G) Immunofluorescence staining to detect IgM deposit in the kidney sections. (H–J) Flow cytometric analysis of CD86 expression on peritoneal macrophages (H) , splenic macrophages (I) , and DCs (J) in all groups of mice. The data are shown as the means ± SEM ( n = 6 mice/group). ** p
Figure Legend Snippet: Treatment effect of anti-CD180 Ab on lupus-symptoms of MRL/ lpr mice. (A) Proteinuria was collected weekly and determined by ELISA. (B) Representative images of marked splenomegaly in all groups of mice. (C,D) ELISA analysis of the anti-dsDNA antibody (C) and anti-RNA antibody (D) in serum. (E) H E staining of the kidney sections from all groups of mice. (F) Immunofluorescence staining to detect IgG deposit in the kidney sections. (G) Immunofluorescence staining to detect IgM deposit in the kidney sections. (H–J) Flow cytometric analysis of CD86 expression on peritoneal macrophages (H) , splenic macrophages (I) , and DCs (J) in all groups of mice. The data are shown as the means ± SEM ( n = 6 mice/group). ** p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Flow Cytometry, Expressing

Treatment effect of anti-CD180 Ab on lupus-symptoms of IMQ-treated mice. (A) Treatment schematic of anti-CD180 Ab and IgG2b injection to mice following epicutaneous application of the TLR7 agonist imiquimod. (B) Proteinuria was collected weekly and determined by ELISA. (C) Representative images of marked splenomegaly in all groups of mice. (D,E) ELISA analysis of the anti-dsDNA antibody (D) and anti-RNA antibody (E) in serum. (F) H E staining of the kidney sections from all groups of mice. (G) Immunofluorescence staining to detect IgG deposit in the kidney sections. (H) Immunofluorescence staining to detect IgM deposit in the kidney sections. (I–K) Flow cytometric analysis of CD86 expression on peritoneal macrophages (I) , splenic macrophages (J) , and DCs (K) in all groups of mice. The data are shown as the means ± SEM ( n = 7 mice/group). * p
Figure Legend Snippet: Treatment effect of anti-CD180 Ab on lupus-symptoms of IMQ-treated mice. (A) Treatment schematic of anti-CD180 Ab and IgG2b injection to mice following epicutaneous application of the TLR7 agonist imiquimod. (B) Proteinuria was collected weekly and determined by ELISA. (C) Representative images of marked splenomegaly in all groups of mice. (D,E) ELISA analysis of the anti-dsDNA antibody (D) and anti-RNA antibody (E) in serum. (F) H E staining of the kidney sections from all groups of mice. (G) Immunofluorescence staining to detect IgG deposit in the kidney sections. (H) Immunofluorescence staining to detect IgM deposit in the kidney sections. (I–K) Flow cytometric analysis of CD86 expression on peritoneal macrophages (I) , splenic macrophages (J) , and DCs (K) in all groups of mice. The data are shown as the means ± SEM ( n = 7 mice/group). * p

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Flow Cytometry, Expressing

4) Product Images from "Complexin 2 regulates secretion of immunoglobulin in antibody‐secreting cells. Complexin 2 regulates secretion of immunoglobulin in antibody‐secreting cells"

Article Title: Complexin 2 regulates secretion of immunoglobulin in antibody‐secreting cells. Complexin 2 regulates secretion of immunoglobulin in antibody‐secreting cells

Journal: Immunity, Inflammation and Disease

doi: 10.1002/iid3.276

Immunophenotype analysis of CPLX2 KO mice. A, Photo of spleens, spleen weight, and H E‐stained spleen sections (20×) of WT and CPLX2 KO mice. H E‐stained sections are one representative image of two independent experiments with two mice per experiment. Scale bars = 500 μm. B, Frequencies of immune cell subsets in spleen were determined by flow cytometry. The gating strategies used to identify the indicated cell subsets are referred to Figure S2A. C, Concentrations of serum immunoglobulin G (IgG) (WT: n = 14, KO: n = 12), IgA (WT: n = 11, KO: n = 11), IgM (WT: n = 21, KO: n = 22), and IgE (WT: n = 10, KO: n = 10) in WT and CPLX2 KO mice were measured by ELISA. D, Serum IgM concentrations were compared between age‐matched male (WT: n = 10, KO: n = 8) and female mice (WT: n = 11, KO: n = 14). A, Data of spleen weight are representative of two independent experiments with two to three mice per experiment. B, Data are pooled from three independent experiments with one to three mice per experiment. C and D, Data are pooled from four independent experiments with two to six mice per experiment. Data are presented as mean ± SEM. CPLX, complexin; ELISA, enzyme‐linked immunosorbent assay; H E, hematoxylin eosin; KO, knockout; N.D., not detected; WT, wild‐type. **** P
Figure Legend Snippet: Immunophenotype analysis of CPLX2 KO mice. A, Photo of spleens, spleen weight, and H E‐stained spleen sections (20×) of WT and CPLX2 KO mice. H E‐stained sections are one representative image of two independent experiments with two mice per experiment. Scale bars = 500 μm. B, Frequencies of immune cell subsets in spleen were determined by flow cytometry. The gating strategies used to identify the indicated cell subsets are referred to Figure S2A. C, Concentrations of serum immunoglobulin G (IgG) (WT: n = 14, KO: n = 12), IgA (WT: n = 11, KO: n = 11), IgM (WT: n = 21, KO: n = 22), and IgE (WT: n = 10, KO: n = 10) in WT and CPLX2 KO mice were measured by ELISA. D, Serum IgM concentrations were compared between age‐matched male (WT: n = 10, KO: n = 8) and female mice (WT: n = 11, KO: n = 14). A, Data of spleen weight are representative of two independent experiments with two to three mice per experiment. B, Data are pooled from three independent experiments with one to three mice per experiment. C and D, Data are pooled from four independent experiments with two to six mice per experiment. Data are presented as mean ± SEM. CPLX, complexin; ELISA, enzyme‐linked immunosorbent assay; H E, hematoxylin eosin; KO, knockout; N.D., not detected; WT, wild‐type. **** P

Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Knock-Out

5) Product Images from "Complexin 2 regulates secretion of immunoglobulin in antibody‐secreting cells. Complexin 2 regulates secretion of immunoglobulin in antibody‐secreting cells"

Article Title: Complexin 2 regulates secretion of immunoglobulin in antibody‐secreting cells. Complexin 2 regulates secretion of immunoglobulin in antibody‐secreting cells

Journal: Immunity, Inflammation and Disease

doi: 10.1002/iid3.276

Immunophenotype analysis of CPLX2 KO mice. A, Photo of spleens, spleen weight, and H E‐stained spleen sections (20×) of WT and CPLX2 KO mice. H E‐stained sections are one representative image of two independent experiments with two mice per experiment. Scale bars = 500 μm. B, Frequencies of immune cell subsets in spleen were determined by flow cytometry. The gating strategies used to identify the indicated cell subsets are referred to Figure S2A. C, Concentrations of serum immunoglobulin G (IgG) (WT: n = 14, KO: n = 12), IgA (WT: n = 11, KO: n = 11), IgM (WT: n = 21, KO: n = 22), and IgE (WT: n = 10, KO: n = 10) in WT and CPLX2 KO mice were measured by ELISA. D, Serum IgM concentrations were compared between age‐matched male (WT: n = 10, KO: n = 8) and female mice (WT: n = 11, KO: n = 14). A, Data of spleen weight are representative of two independent experiments with two to three mice per experiment. B, Data are pooled from three independent experiments with one to three mice per experiment. C and D, Data are pooled from four independent experiments with two to six mice per experiment. Data are presented as mean ± SEM. CPLX, complexin; ELISA, enzyme‐linked immunosorbent assay; H E, hematoxylin eosin; KO, knockout; N.D., not detected; WT, wild‐type. **** P
Figure Legend Snippet: Immunophenotype analysis of CPLX2 KO mice. A, Photo of spleens, spleen weight, and H E‐stained spleen sections (20×) of WT and CPLX2 KO mice. H E‐stained sections are one representative image of two independent experiments with two mice per experiment. Scale bars = 500 μm. B, Frequencies of immune cell subsets in spleen were determined by flow cytometry. The gating strategies used to identify the indicated cell subsets are referred to Figure S2A. C, Concentrations of serum immunoglobulin G (IgG) (WT: n = 14, KO: n = 12), IgA (WT: n = 11, KO: n = 11), IgM (WT: n = 21, KO: n = 22), and IgE (WT: n = 10, KO: n = 10) in WT and CPLX2 KO mice were measured by ELISA. D, Serum IgM concentrations were compared between age‐matched male (WT: n = 10, KO: n = 8) and female mice (WT: n = 11, KO: n = 14). A, Data of spleen weight are representative of two independent experiments with two to three mice per experiment. B, Data are pooled from three independent experiments with one to three mice per experiment. C and D, Data are pooled from four independent experiments with two to six mice per experiment. Data are presented as mean ± SEM. CPLX, complexin; ELISA, enzyme‐linked immunosorbent assay; H E, hematoxylin eosin; KO, knockout; N.D., not detected; WT, wild‐type. **** P

Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Knock-Out

6) Product Images from "Utility of Humanized BLT Mice for Analysis of Dengue Virus Infection and Antiviral Drug Testing"

Article Title: Utility of Humanized BLT Mice for Analysis of Dengue Virus Infection and Antiviral Drug Testing

Journal: Journal of Virology

doi: 10.1128/JVI.03085-13

Human humoral immune responses in BLT-NOD/ SCID mice infected with DENV-2. (A) Total human IgM antibodies circulating in the bloodstream of DENV-2 Col-infected and control animals were measured by ELISA at days 7, 13, 25, and 32 postinfection. Black diamonds
Figure Legend Snippet: Human humoral immune responses in BLT-NOD/ SCID mice infected with DENV-2. (A) Total human IgM antibodies circulating in the bloodstream of DENV-2 Col-infected and control animals were measured by ELISA at days 7, 13, 25, and 32 postinfection. Black diamonds

Techniques Used: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

7) Product Images from "Heat Shock Protein-based therapy as a potential candidate for treating sphingolipidoses"

Article Title: Heat Shock Protein-based therapy as a potential candidate for treating sphingolipidoses

Journal: Science translational medicine

doi: 10.1126/scitranslmed.aad9823

Effects of rHsp70 treatment in vitro ( A ) Analysis of rHSP70’s effect on the lipid cofactor Bis(monoacyl)glycerophosphate (BMP)-binding interactions of sphingolipid-catabolic enzymes (GLA, NEU1, ARSA, GLB1, HEXA and HEXB). Data points and association curves (non-linear regression, assuming one-phase association) are depicted on all graphs for rHSP70 except for HEXB (no curve-fit). For GLA, NEU1 and ARSA the effect of the Trp90Phe point mutation in HSP70 (W90F) lacking the capacity to interact with BMP was also analysed. Only for NEU1 could a one-phase association curve be fitted. For ARSA and GLA the signal was too weak to establish any meaningful regression (linear regression shown as a guide). ( B ) Quantification of lysosomal area in confocal cross sections of primary fibroblasts from patients with different lysosomal storage diseases, either sham-treated (Vehicle) or treated for 24h with 300nM rHSP70. The representative microscopic images on the right show the effect of 24h rHSP70 (green) treatment on the volume of the lysosomal compartment (red) in fibroblasts from a patient with Farber disease. White dotted lines indicate the positions of cells with endocytosed rHSP70. Scale bar 50μm. ( C ) Quantification of lysosomal area of confocal cross sections of primary patient fibroblasts from patients with Niemann-Pick type C disease, either sham-treated (Vehicle) or treated for 24h with 300nM rHSP70. ( D ) Quantification of lysosomal area of confocal cross sections of primary patient fibroblasts from two Niemann-Pick type A patients treated with 300nM of the indicated recombinant proteins for 24h. ( A ) The individual values represent the mean of three independent experiments, P ≤ 0.05. ( B-D ) All values represent mean ± S.D. for a minimum of three independent experiments. A minimum of 100 cells were analyzed for each independent experiment P ≤ 0.05. Two-sample comparisons were performed by employing Student’s t-test, multiple comparisons were analysed by one-way ANOVA followed by Dunnet’s multiple comparison test. BMP: Bis(monoacyl)glycerophosphate, GLA: alpha-Galactosidase A, NEU1: Neuraminidase 1, ARSA: Arylsulfatase, GLB1: beta-galactosidase, HEXA: Hexosaminidase A, HEXB: Hexosaminidase B
Figure Legend Snippet: Effects of rHsp70 treatment in vitro ( A ) Analysis of rHSP70’s effect on the lipid cofactor Bis(monoacyl)glycerophosphate (BMP)-binding interactions of sphingolipid-catabolic enzymes (GLA, NEU1, ARSA, GLB1, HEXA and HEXB). Data points and association curves (non-linear regression, assuming one-phase association) are depicted on all graphs for rHSP70 except for HEXB (no curve-fit). For GLA, NEU1 and ARSA the effect of the Trp90Phe point mutation in HSP70 (W90F) lacking the capacity to interact with BMP was also analysed. Only for NEU1 could a one-phase association curve be fitted. For ARSA and GLA the signal was too weak to establish any meaningful regression (linear regression shown as a guide). ( B ) Quantification of lysosomal area in confocal cross sections of primary fibroblasts from patients with different lysosomal storage diseases, either sham-treated (Vehicle) or treated for 24h with 300nM rHSP70. The representative microscopic images on the right show the effect of 24h rHSP70 (green) treatment on the volume of the lysosomal compartment (red) in fibroblasts from a patient with Farber disease. White dotted lines indicate the positions of cells with endocytosed rHSP70. Scale bar 50μm. ( C ) Quantification of lysosomal area of confocal cross sections of primary patient fibroblasts from patients with Niemann-Pick type C disease, either sham-treated (Vehicle) or treated for 24h with 300nM rHSP70. ( D ) Quantification of lysosomal area of confocal cross sections of primary patient fibroblasts from two Niemann-Pick type A patients treated with 300nM of the indicated recombinant proteins for 24h. ( A ) The individual values represent the mean of three independent experiments, P ≤ 0.05. ( B-D ) All values represent mean ± S.D. for a minimum of three independent experiments. A minimum of 100 cells were analyzed for each independent experiment P ≤ 0.05. Two-sample comparisons were performed by employing Student’s t-test, multiple comparisons were analysed by one-way ANOVA followed by Dunnet’s multiple comparison test. BMP: Bis(monoacyl)glycerophosphate, GLA: alpha-Galactosidase A, NEU1: Neuraminidase 1, ARSA: Arylsulfatase, GLB1: beta-galactosidase, HEXA: Hexosaminidase A, HEXB: Hexosaminidase B

Techniques Used: In Vitro, Binding Assay, Mutagenesis, Recombinant

8) Product Images from "Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals"

Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01364

Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P
Figure Legend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P

Techniques Used: Activity Assay, Indirect Immunoperoxidase Assay, Software, Activation Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Mouse Assay

TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P
Figure Legend Snippet: TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P

Techniques Used: Labeling, Enzyme-linked Immunosorbent Assay, Cell Culture, Mouse Assay, Plasmid Preparation

Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P
Figure Legend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P

Techniques Used: Mouse Assay, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P
Figure Legend Snippet: TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P

Techniques Used: Activation Assay, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Expressing, Mouse Assay, Plasmid Preparation, Injection

9) Product Images from "Human Vγ9Vδ2-T Cells Synergize CD4+ T Follicular Helper Cells to Produce Influenza Virus-Specific Antibody"

Article Title: Human Vγ9Vδ2-T Cells Synergize CD4+ T Follicular Helper Cells to Produce Influenza Virus-Specific Antibody

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00599

Vγ9Vδ2-T cells increased H9N2 virus-specific IgG and IgM productions in vivo . Specific immune cells-depleted and whole peripheral blood mononuclear cells (PBMC)-humanized mice were vaccinated with UV-inactivated H9N2 virus through i.p. pathway on day 0 and 7. Serum was collected on day 21 post the first vaccination. (A,B) Total IgG and IgM in serum (C,D) H9N2 virus-specific IgG and IgM in serum that treated with receptor destroyed enzyme (RDE) were determined by enzyme-linked immunosorbent assay. (E) H9N2-specific antibodies in serum that treated with RDE were determined by hemagglutination inhibition assay. The data shown were the represent of four independent experiments. (F) Representative histological analysis, immunohistological stainings for human CD4 T, B, and γδ-T cells in formalin-fixed paraffin-embedded spleen in humanized mice constructed with whole PBMCs after UV-H9N2 virus immunization. Scale bars = 100 μm. Bottom panels showed the higher-magnification views of the respective boxed areas in the top panels. Scale bars = 50 μm. The data shown are the mean ± SEM. * p
Figure Legend Snippet: Vγ9Vδ2-T cells increased H9N2 virus-specific IgG and IgM productions in vivo . Specific immune cells-depleted and whole peripheral blood mononuclear cells (PBMC)-humanized mice were vaccinated with UV-inactivated H9N2 virus through i.p. pathway on day 0 and 7. Serum was collected on day 21 post the first vaccination. (A,B) Total IgG and IgM in serum (C,D) H9N2 virus-specific IgG and IgM in serum that treated with receptor destroyed enzyme (RDE) were determined by enzyme-linked immunosorbent assay. (E) H9N2-specific antibodies in serum that treated with RDE were determined by hemagglutination inhibition assay. The data shown were the represent of four independent experiments. (F) Representative histological analysis, immunohistological stainings for human CD4 T, B, and γδ-T cells in formalin-fixed paraffin-embedded spleen in humanized mice constructed with whole PBMCs after UV-H9N2 virus immunization. Scale bars = 100 μm. Bottom panels showed the higher-magnification views of the respective boxed areas in the top panels. Scale bars = 50 μm. The data shown are the mean ± SEM. * p

Techniques Used: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, HI Assay, Formalin-fixed Paraffin-Embedded, Construct

Vγ9Vδ2-T cells facilitated influenza virus-specific antibody production. Naïve CD4 T cells were cultured with UV-H9N2 virus-pulsed dendritic cells for 4–5 days, then, the activated CD4 T cells were cultured with CD19 + B cells from same donor with or without Vγ9Vδ2-T cells for another 7–10 days. (A,B) Total IgG and IgM in supernatant on day 7 were detected by enzyme-linked immunosorbent assay (ELISA). (C,D) H9N2 virus-specific IgG and IgM were detected by ELISA on day 7. Each dot means one donor. The data shown are the mean ± SEM. * p
Figure Legend Snippet: Vγ9Vδ2-T cells facilitated influenza virus-specific antibody production. Naïve CD4 T cells were cultured with UV-H9N2 virus-pulsed dendritic cells for 4–5 days, then, the activated CD4 T cells were cultured with CD19 + B cells from same donor with or without Vγ9Vδ2-T cells for another 7–10 days. (A,B) Total IgG and IgM in supernatant on day 7 were detected by enzyme-linked immunosorbent assay (ELISA). (C,D) H9N2 virus-specific IgG and IgM were detected by ELISA on day 7. Each dot means one donor. The data shown are the mean ± SEM. * p

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

10) Product Images from "Collagen‐derived peptides modulate CD4+ T‐cell differentiation and suppress allergic responses in mice"

Article Title: Collagen‐derived peptides modulate CD4+ T‐cell differentiation and suppress allergic responses in mice

Journal: Immunity, Inflammation and Disease

doi: 10.1002/iid3.213

Effects of collagen‐peptide administration on humoral immune responses. BALB/c mice fed a diet with or without collagen peptides were intraperitoneally injected with 10 μg of OVA and Alum at weeks 1, 2, and 3. Serum was collected from each mouse, and total and OVA‐specific IgE and IgG were measured using ELISA. Values are means ± SEM ( n = 16) obtained by two independent experiments. ** P
Figure Legend Snippet: Effects of collagen‐peptide administration on humoral immune responses. BALB/c mice fed a diet with or without collagen peptides were intraperitoneally injected with 10 μg of OVA and Alum at weeks 1, 2, and 3. Serum was collected from each mouse, and total and OVA‐specific IgE and IgG were measured using ELISA. Values are means ± SEM ( n = 16) obtained by two independent experiments. ** P

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

11) Product Images from "Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals"

Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01364

Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P
Figure Legend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P

Techniques Used: Activity Assay, Indirect Immunoperoxidase Assay, Software, Activation Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Mouse Assay

TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P
Figure Legend Snippet: TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P

Techniques Used: Labeling, Enzyme-linked Immunosorbent Assay, Cell Culture, Mouse Assay, Plasmid Preparation

Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P
Figure Legend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P

Techniques Used: Mouse Assay, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P
Figure Legend Snippet: TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P

Techniques Used: Activation Assay, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Expressing, Mouse Assay, Plasmid Preparation, Injection

12) Product Images from "Secreted IgD Amplifies Humoral T Helper-2 Responses by Binding Basophils Via Galectin-9 and CD44"

Article Title: Secreted IgD Amplifies Humoral T Helper-2 Responses by Binding Basophils Via Galectin-9 and CD44

Journal: Immunity

doi: 10.1016/j.immuni.2018.08.013

IgD Ligation by Antigen Elicits Basophil Activation (A) ELISA of serum IgD from WT Balb/c (n=29), WT C57BL/6 (n=15), Ighd −/− Balb/c (n=20) or Ighd −/− C57BL/6 (n=10) mice. (B) Left: FCM of CD49b and IgE on basophils (black gate) from circulating CD45 + NTNB cells of a WT Balb/c mouse. NTNB, non-T non-B. Right: FCM of IgD on circulating basophils (blue open profile) from a WT Balb/c mouse. Ctrl, isotype-matched control (gray solid profile). (C) FCM of IgD + basophils from peripheral blood, spleen, bone marrow (BM), lung or mesenteric lymph nodes (MLNs) of WT Balb/c mice (n=5). (D) Imaging FCM of IgD, FcεRI and CD49b from a representative viable Ghost Dye Violet 510 (GV510) − splenic basophil of a WT Balb/c mouse. Scale bar, 5 μm. (E) FCM quantification of FcεRI + CD49b + basophils from BM, blood, spleen or lung CD45 + NTNB cells of WT Balb/c (n=10) or Ighd −/− (n=10) mice. (F) FCM of Il4 -driven GFP expression in splenic or lung FcεRI + CD49b + basophils from WT Balb/c Il4 GFP (n=5) or Il4 GFP Ighd −/− (n=5) mice. MFI, mean fluorescence intensity. (G) ELISA of IL-4 and IL-13 from serum of WT Balb/c or Ighd −/− mice (n=10). (H) Schematics of i.v. reconstitution μMT or Rag2 −/− mice with NP-reactive IgD (NP-IgD) followed by i.p. injection of anti-IgD or i.n. inoculation of NP-OVA. (I) FCM of IgD on splenic FcεRI + CD49b + basophils from a μMT mouse before (ctrl) or after reconstitution as in (H). PBS, control phosphate buffer solution (PBS). (J, K) FCM quantification of total, CD200R3 + or IL-4 + basophils from the spleen of μMT C57BL/6 mice (n=10) (J) or the lungs of IgD-deficient Rag2 −/− (n=10) C57BL/6 mice (K) treated as in (H). (L) FCM quantification of total, IgD + , IgE + or IL-4 + basophils from the lungs of WT Balb/c (n=5) or Ighd −/− (n=5) mice 5 d following i.n. exposure to PBS or recombinant TSLP. (M) ELISA of serum IgD from Balb/c mice following i.v. injection of control empty or TSLP-encoding plasmids (n=10). .
Figure Legend Snippet: IgD Ligation by Antigen Elicits Basophil Activation (A) ELISA of serum IgD from WT Balb/c (n=29), WT C57BL/6 (n=15), Ighd −/− Balb/c (n=20) or Ighd −/− C57BL/6 (n=10) mice. (B) Left: FCM of CD49b and IgE on basophils (black gate) from circulating CD45 + NTNB cells of a WT Balb/c mouse. NTNB, non-T non-B. Right: FCM of IgD on circulating basophils (blue open profile) from a WT Balb/c mouse. Ctrl, isotype-matched control (gray solid profile). (C) FCM of IgD + basophils from peripheral blood, spleen, bone marrow (BM), lung or mesenteric lymph nodes (MLNs) of WT Balb/c mice (n=5). (D) Imaging FCM of IgD, FcεRI and CD49b from a representative viable Ghost Dye Violet 510 (GV510) − splenic basophil of a WT Balb/c mouse. Scale bar, 5 μm. (E) FCM quantification of FcεRI + CD49b + basophils from BM, blood, spleen or lung CD45 + NTNB cells of WT Balb/c (n=10) or Ighd −/− (n=10) mice. (F) FCM of Il4 -driven GFP expression in splenic or lung FcεRI + CD49b + basophils from WT Balb/c Il4 GFP (n=5) or Il4 GFP Ighd −/− (n=5) mice. MFI, mean fluorescence intensity. (G) ELISA of IL-4 and IL-13 from serum of WT Balb/c or Ighd −/− mice (n=10). (H) Schematics of i.v. reconstitution μMT or Rag2 −/− mice with NP-reactive IgD (NP-IgD) followed by i.p. injection of anti-IgD or i.n. inoculation of NP-OVA. (I) FCM of IgD on splenic FcεRI + CD49b + basophils from a μMT mouse before (ctrl) or after reconstitution as in (H). PBS, control phosphate buffer solution (PBS). (J, K) FCM quantification of total, CD200R3 + or IL-4 + basophils from the spleen of μMT C57BL/6 mice (n=10) (J) or the lungs of IgD-deficient Rag2 −/− (n=10) C57BL/6 mice (K) treated as in (H). (L) FCM quantification of total, IgD + , IgE + or IL-4 + basophils from the lungs of WT Balb/c (n=5) or Ighd −/− (n=5) mice 5 d following i.n. exposure to PBS or recombinant TSLP. (M) ELISA of serum IgD from Balb/c mice following i.v. injection of control empty or TSLP-encoding plasmids (n=10). .

Techniques Used: Ligation, Activation Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay, Imaging, Expressing, Fluorescence, Injection, Recombinant

Basophil-Bound IgD Interacts with Galectin-9 and Its Ligation Induces Th2 Cell-Associated Cytokine Expression But Inhibits Cytoskeleton Remodeling (A) Schematics of i.p. anti-IgD treated mice i.v. reconstituted or not with secreted IgD. (B-C) ELISA of serum IgG1, IgE (B), IL-4 and IL-13 (C) from WT Balb/c (n=10) or Ighd −/− (n=10) mice in the presence or absence of i.v. reconstitution with secreted IgD (NP-IgD), followed by i.p. injection of anti-IgD. Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to onset of anti-IgD treatment (day 0). (D) IFA of human tonsillar tissue stained for IgD (green), IgM (red), cytokeratin (purple) and nuclei (blue). EP, epithelium; FO, follicle; PC, plasma cell. Original magnification, ×20 (top) or ×63 (bottom). Scale bars, 50 μm (top) and 5 μm (bottom). (E) ELISA of IgD specific to α-s-casein, β-lactoglobulin (β-LGB) or α-lactalbumin (α-LAL) from plasma of FPIES children with (n=5) or without (n=5) dietary milk restrictions. (F) FCM of IgD (red open profile) bound to human tonsillar or circulating CD123 + FcεRI + basophils (red gate). Gray open profiles, control isotype-matched antibody with irrelevant binding activity. (G) Microarray analysis of genes expressed by human basophils upon IgD cross-linking. The Volcano plot represents genes differentially expressed by basophils treated with anti-IgD or a F(ab’)2 control antibody (ctrl). Red and blue dots, up-regulated and down-regulated genes, respectively; FC, fold change. (H) Heat map of coordinated gene sets identified by gene set enrichment analysis in human basophils treated as in (G). NES (normalized enrichment score) indicates correlation between individual gene sets. Positive correlation, NES > 0 (yellow gradient); negative correlation, NES
Figure Legend Snippet: Basophil-Bound IgD Interacts with Galectin-9 and Its Ligation Induces Th2 Cell-Associated Cytokine Expression But Inhibits Cytoskeleton Remodeling (A) Schematics of i.p. anti-IgD treated mice i.v. reconstituted or not with secreted IgD. (B-C) ELISA of serum IgG1, IgE (B), IL-4 and IL-13 (C) from WT Balb/c (n=10) or Ighd −/− (n=10) mice in the presence or absence of i.v. reconstitution with secreted IgD (NP-IgD), followed by i.p. injection of anti-IgD. Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to onset of anti-IgD treatment (day 0). (D) IFA of human tonsillar tissue stained for IgD (green), IgM (red), cytokeratin (purple) and nuclei (blue). EP, epithelium; FO, follicle; PC, plasma cell. Original magnification, ×20 (top) or ×63 (bottom). Scale bars, 50 μm (top) and 5 μm (bottom). (E) ELISA of IgD specific to α-s-casein, β-lactoglobulin (β-LGB) or α-lactalbumin (α-LAL) from plasma of FPIES children with (n=5) or without (n=5) dietary milk restrictions. (F) FCM of IgD (red open profile) bound to human tonsillar or circulating CD123 + FcεRI + basophils (red gate). Gray open profiles, control isotype-matched antibody with irrelevant binding activity. (G) Microarray analysis of genes expressed by human basophils upon IgD cross-linking. The Volcano plot represents genes differentially expressed by basophils treated with anti-IgD or a F(ab’)2 control antibody (ctrl). Red and blue dots, up-regulated and down-regulated genes, respectively; FC, fold change. (H) Heat map of coordinated gene sets identified by gene set enrichment analysis in human basophils treated as in (G). NES (normalized enrichment score) indicates correlation between individual gene sets. Positive correlation, NES > 0 (yellow gradient); negative correlation, NES

Techniques Used: Ligation, Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection, Concentration Assay, Immunofluorescence, Staining, Binding Assay, Activity Assay, Microarray

IgD Binds to Basophils through Galectin-9 and CD44 (A) IB of galectin-9 following IP of human IgD and galectin-9 protein mix with control (ctrl) or anti-IgD antibodies. Prior to IP, the protein mix was supplemented with control PBS, glucose or lactose. (B) FCM of human IgD on human KU812 cells cultured for 30 min with control medium alone (ctrl), IgD or an IgD-galectin-9 complex formed by pre-incubating IgD with galectin-9 for 10 min. (C) FCM of human IgD or galectin-9 on KU812 cells cultured with IgD-galectin-9 in the presence of medium alone (ctrl), glucose or lactose for 30 min. (D) FCM of human IgD or CD44 on KU812 cells treated with scrambled (ctrl) or CD44-targeting small interfering RNA (siRNA) and later incubated with or without (ctrl) IgD-galectin-9. MFI, mean fluorescence intensity. (E) Confocal imaging of human basophils stained for IgD (blue), galectin-9 (green) and CD44 (red). Scale bar, 0.5 μm. (F) FCM of IgD on human basophils incubated with or without (ctrl) IgD-galectin-9 for 30 min. (G) ELISA of IL-4 from human basophils incubated with medium alone (ctrl) and with or without the IgD-galectin-9 complex in the presence or absence of IgD cross-linking by anti-IgD for 18 h. (H, I) FCM of IgD + basophils from the spleen or lung of WT C57BL/6, Lgals9 −/− (H) or Cd44 −/− (I) mice. (J, K) ELISA of serum IgG1 and IgE to NP from WT Balb/c (n=10), Lgals9 −/− (n=8) (J) or Cd44 −/− (n=10) (K) mice following s.c. immunization with NP-OVA and papain. Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to the onset of anti-IgD treatment (day 0). (L) ELISA of serum total IgG1 and IgE from WT C57BL/6 controls (n=10) and Lgals9 −/− mice (n=8) following i.p. injection of anti-IgD. .
Figure Legend Snippet: IgD Binds to Basophils through Galectin-9 and CD44 (A) IB of galectin-9 following IP of human IgD and galectin-9 protein mix with control (ctrl) or anti-IgD antibodies. Prior to IP, the protein mix was supplemented with control PBS, glucose or lactose. (B) FCM of human IgD on human KU812 cells cultured for 30 min with control medium alone (ctrl), IgD or an IgD-galectin-9 complex formed by pre-incubating IgD with galectin-9 for 10 min. (C) FCM of human IgD or galectin-9 on KU812 cells cultured with IgD-galectin-9 in the presence of medium alone (ctrl), glucose or lactose for 30 min. (D) FCM of human IgD or CD44 on KU812 cells treated with scrambled (ctrl) or CD44-targeting small interfering RNA (siRNA) and later incubated with or without (ctrl) IgD-galectin-9. MFI, mean fluorescence intensity. (E) Confocal imaging of human basophils stained for IgD (blue), galectin-9 (green) and CD44 (red). Scale bar, 0.5 μm. (F) FCM of IgD on human basophils incubated with or without (ctrl) IgD-galectin-9 for 30 min. (G) ELISA of IL-4 from human basophils incubated with medium alone (ctrl) and with or without the IgD-galectin-9 complex in the presence or absence of IgD cross-linking by anti-IgD for 18 h. (H, I) FCM of IgD + basophils from the spleen or lung of WT C57BL/6, Lgals9 −/− (H) or Cd44 −/− (I) mice. (J, K) ELISA of serum IgG1 and IgE to NP from WT Balb/c (n=10), Lgals9 −/− (n=8) (J) or Cd44 −/− (n=10) (K) mice following s.c. immunization with NP-OVA and papain. Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to the onset of anti-IgD treatment (day 0). (L) ELISA of serum total IgG1 and IgE from WT C57BL/6 controls (n=10) and Lgals9 −/− mice (n=8) following i.p. injection of anti-IgD. .

Techniques Used: Cell Culture, Small Interfering RNA, Incubation, Fluorescence, Imaging, Staining, Enzyme-linked Immunosorbent Assay, Mouse Assay, Concentration Assay, Injection

Ligation of Basophil-Bound IgD by Antigen Enhances IgG1 and IgE Responses (A) Schematics of i.p. immunization with NP-OVA and papain. (B) ELISA of serum OVA-specific IgG1 and IgE from WT Balb/c (n=10) or Ighd −/− (n=10) mice immunized as in (A). Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to the onset of immunization (day 0). (C-D) FCM quantification of total and IL-4-expressing splenic PD-1 high CXCR5 high Tfh cells from WT Balb/c (n=5) or Ighd −/− (n=5) mice immunized as in (A). (E, F) ELISA of serum NP-specific IgG1 from WT Balb/c (n=10) or Ighd −/− (n=10) mice immunized as in (A). BSA haptenated with 4 or 16 NPs was used to measure high-affinity (HA) and both HA and lowaffinity (LA) IgG1, respectively. (G) IFA of splenic tissue from immunized Balb/c mice stained for IgD (green), IgM (red) and nuclei (blue) following i.p. immunization as in (B). Inset: IgD + IgM − plasmablast next to IgD − IgM + plasmablast. FO, follicle. Original magnification, ×10 with ×2 enlargement. Scale bar, 50 μm. (H) ELISPOT of spleen ASCs expressing NP-specific IgD from WT Balb/c (n=5) or Ighd −/− (n=5) mice 3 d following i.p. immunization with PBS or NP-OVA and papain. (I) Schematics of i.v. reconstitution with NP-reactive IgD (NP-IgD) followed by i.p. immunization with NP-OVA and papain. (J) ELISA of serum OVA-specific IgG1 and IgE from WT Balb/c controls (n=10), Ighd −/− mice (n=10) or NP-IgD-reconstituted Ighd −/− mice (n=10) following i.p. immunization with NP-OVA and papain as in (I). (K) FCM of circulating FcεRI + IgE + basophils from WT Balb/c mice after i.v. injection of a control (ctrl) IgG2b antibody or a basophil-depleting anti-CD200R3 antibody. (L) ELISA of serum total IgD as well as serum NP-specific IgG1 and IgE in control (n=10) or basophil-depleted (n=10) WT Balb/c mice after i.p. immunization with NP-OVA and papain. .
Figure Legend Snippet: Ligation of Basophil-Bound IgD by Antigen Enhances IgG1 and IgE Responses (A) Schematics of i.p. immunization with NP-OVA and papain. (B) ELISA of serum OVA-specific IgG1 and IgE from WT Balb/c (n=10) or Ighd −/− (n=10) mice immunized as in (A). Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to the onset of immunization (day 0). (C-D) FCM quantification of total and IL-4-expressing splenic PD-1 high CXCR5 high Tfh cells from WT Balb/c (n=5) or Ighd −/− (n=5) mice immunized as in (A). (E, F) ELISA of serum NP-specific IgG1 from WT Balb/c (n=10) or Ighd −/− (n=10) mice immunized as in (A). BSA haptenated with 4 or 16 NPs was used to measure high-affinity (HA) and both HA and lowaffinity (LA) IgG1, respectively. (G) IFA of splenic tissue from immunized Balb/c mice stained for IgD (green), IgM (red) and nuclei (blue) following i.p. immunization as in (B). Inset: IgD + IgM − plasmablast next to IgD − IgM + plasmablast. FO, follicle. Original magnification, ×10 with ×2 enlargement. Scale bar, 50 μm. (H) ELISPOT of spleen ASCs expressing NP-specific IgD from WT Balb/c (n=5) or Ighd −/− (n=5) mice 3 d following i.p. immunization with PBS or NP-OVA and papain. (I) Schematics of i.v. reconstitution with NP-reactive IgD (NP-IgD) followed by i.p. immunization with NP-OVA and papain. (J) ELISA of serum OVA-specific IgG1 and IgE from WT Balb/c controls (n=10), Ighd −/− mice (n=10) or NP-IgD-reconstituted Ighd −/− mice (n=10) following i.p. immunization with NP-OVA and papain as in (I). (K) FCM of circulating FcεRI + IgE + basophils from WT Balb/c mice after i.v. injection of a control (ctrl) IgG2b antibody or a basophil-depleting anti-CD200R3 antibody. (L) ELISA of serum total IgD as well as serum NP-specific IgG1 and IgE in control (n=10) or basophil-depleted (n=10) WT Balb/c mice after i.p. immunization with NP-OVA and papain. .

Techniques Used: Ligation, Enzyme-linked Immunosorbent Assay, Mouse Assay, Concentration Assay, Expressing, Immunofluorescence, Staining, Enzyme-linked Immunospot, Injection

IgD Ligation by Antigen Induces Basophil Expression of IL-4 (A) Schematics of s.c. immunization with NP-OVA combined with control PBS, papain or NP-IgD. (B) Confocal imaging of CD169 (subcapsular sinus macrophage molecule, red), B220 (B cell molecule, blue) and Mcpt8 (YFP, yellow) from draining lymph node (DLN) of a C57BL/6 Mcpt8 YFP mouse immunized for 6 h as in (A). FO, follicle; dashed line, follicular border. Original magnification, ×5; rightbottom panel, ×40. Scale bars, 50 μm (top and bottom-left panels) or 5 μm (bottom-right panel). (C-E) FCM quantification of total YFP + or GFP + basophils and qRT-PCR quantification of Il4 transcripts encoding IL-4 from the DLN of C57BL/6 Mcpt8 YFP (n=5) mice (C, D) or Balb/c Il4 GFP (n=10) mice (E) 6 h following s.c. immunization as in (A). qRT-PCR results (D, bottom graph) are presented as relative expression (RE) compared to mRNA for glyceraldeheyde phosphate dehydrogenase (GAPDH). (F) Schematics of s.c. immunization with NP-OVA combined with papain, alum or CFA. (G) ELISA of serum NP-specific IgD from WT Balb/c mice (n=17) following s.c. immunization with NP-OVA and papain. PI, pre-immune (day −1 relatively to the onset of immunization (day 0). (H) ELISA of serum NP-specific IgG1 and IgE from WT Balb/c (n=12) or Ighd −/− (n=16) mice following s.c. immunization with NP-OVA and papain. (I) ELISA of serum NP-specific IgG1 and IgE from WT Balb/c (n=15) or Ighd −/− (n=15) mice following s.c. immunization with NP-OVA and alum. (J) ELISA of serum NP-specific IgG2a and IgG2b from Balb/c (n=10) or Ighd −/− (n=10) mice following s.c. immunization with NP-OVA and CFA. Data show one experiment of three with similar results (B, C) or summarize results from two experiments with 5–10 (D, E) or 10–17 (G-J) mice per experimental group. Results are presented as mean ± SEM; *p
Figure Legend Snippet: IgD Ligation by Antigen Induces Basophil Expression of IL-4 (A) Schematics of s.c. immunization with NP-OVA combined with control PBS, papain or NP-IgD. (B) Confocal imaging of CD169 (subcapsular sinus macrophage molecule, red), B220 (B cell molecule, blue) and Mcpt8 (YFP, yellow) from draining lymph node (DLN) of a C57BL/6 Mcpt8 YFP mouse immunized for 6 h as in (A). FO, follicle; dashed line, follicular border. Original magnification, ×5; rightbottom panel, ×40. Scale bars, 50 μm (top and bottom-left panels) or 5 μm (bottom-right panel). (C-E) FCM quantification of total YFP + or GFP + basophils and qRT-PCR quantification of Il4 transcripts encoding IL-4 from the DLN of C57BL/6 Mcpt8 YFP (n=5) mice (C, D) or Balb/c Il4 GFP (n=10) mice (E) 6 h following s.c. immunization as in (A). qRT-PCR results (D, bottom graph) are presented as relative expression (RE) compared to mRNA for glyceraldeheyde phosphate dehydrogenase (GAPDH). (F) Schematics of s.c. immunization with NP-OVA combined with papain, alum or CFA. (G) ELISA of serum NP-specific IgD from WT Balb/c mice (n=17) following s.c. immunization with NP-OVA and papain. PI, pre-immune (day −1 relatively to the onset of immunization (day 0). (H) ELISA of serum NP-specific IgG1 and IgE from WT Balb/c (n=12) or Ighd −/− (n=16) mice following s.c. immunization with NP-OVA and papain. (I) ELISA of serum NP-specific IgG1 and IgE from WT Balb/c (n=15) or Ighd −/− (n=15) mice following s.c. immunization with NP-OVA and alum. (J) ELISA of serum NP-specific IgG2a and IgG2b from Balb/c (n=10) or Ighd −/− (n=10) mice following s.c. immunization with NP-OVA and CFA. Data show one experiment of three with similar results (B, C) or summarize results from two experiments with 5–10 (D, E) or 10–17 (G-J) mice per experimental group. Results are presented as mean ± SEM; *p

Techniques Used: Ligation, Expressing, Imaging, Quantitative RT-PCR, Mouse Assay, Enzyme-linked Immunosorbent Assay

IgD Attenuates Acute Lung Inflammation Following Secondary Antigen Exposure (A) Schematics of i.p. OVA sensitization followed by a challenge consisting of four consecutive i.t. inoculations of OVA or control PBS. (B) ELISA of OVA-specific IgD and IgE from serum of WT Balb/c (n=10) or Ighd −/− (n=10) mice treated as in (A). Dashed line, maximum antibody concentration 4 hs after the last i.t. inoculation of PBS. (C) FCM quantitation of IgE on l basophils from lungs of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). (D) Microscopic quantitation of total cells from the bronchoalveolar lavage (BAL) of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). (E, F) FCM quantitation of CD49b + IgE + basophils from lungs of WT Balb/c (n=10) or Ighd −/− (n=10) mice treated as in (A). NTNB, non-T non-B. (G) ELISA of Mcpt8 from serum of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). (H, I) FCM quantitation of CD45 + Siglec-F + eosinophils from lungs of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). Data summarize two experiments with at least five mice per experimental group. Results are presented as mean ± SEM; *p
Figure Legend Snippet: IgD Attenuates Acute Lung Inflammation Following Secondary Antigen Exposure (A) Schematics of i.p. OVA sensitization followed by a challenge consisting of four consecutive i.t. inoculations of OVA or control PBS. (B) ELISA of OVA-specific IgD and IgE from serum of WT Balb/c (n=10) or Ighd −/− (n=10) mice treated as in (A). Dashed line, maximum antibody concentration 4 hs after the last i.t. inoculation of PBS. (C) FCM quantitation of IgE on l basophils from lungs of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). (D) Microscopic quantitation of total cells from the bronchoalveolar lavage (BAL) of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). (E, F) FCM quantitation of CD49b + IgE + basophils from lungs of WT Balb/c (n=10) or Ighd −/− (n=10) mice treated as in (A). NTNB, non-T non-B. (G) ELISA of Mcpt8 from serum of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). (H, I) FCM quantitation of CD45 + Siglec-F + eosinophils from lungs of WT Balb/c (n=5) or Ighd −/− (n=5) mice treated as in (A). Data summarize two experiments with at least five mice per experimental group. Results are presented as mean ± SEM; *p

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Concentration Assay, Quantitation Assay

13) Product Images from "Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals"

Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01364

Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P
Figure Legend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P

Techniques Used: Activity Assay, Indirect Immunoperoxidase Assay, Software, Activation Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Mouse Assay

TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P
Figure Legend Snippet: TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P

Techniques Used: Labeling, Enzyme-linked Immunosorbent Assay, Cell Culture, Mouse Assay, Plasmid Preparation

Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P
Figure Legend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P

Techniques Used: Mouse Assay, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P
Figure Legend Snippet: TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P

Techniques Used: Activation Assay, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Expressing, Mouse Assay, Plasmid Preparation, Injection

14) Product Images from "A benzenediamine derivate FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting myeloid dendritic cell-secreted BAFF"

Article Title: A benzenediamine derivate FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting myeloid dendritic cell-secreted BAFF

Journal: Acta Biochimica et Biophysica Sinica

doi: 10.1093/abbs/gmw017

FC-99 reduces the percentage, activation, and maturation of B cells in MRL/ lpr mice Serum levels of total IgG (A), IgG2a (B), and IgM (C) were determined by ELISA. (D) The percentage of B220 + B cells in spleens was analyzed by flow cytometry. (E and F)
Figure Legend Snippet: FC-99 reduces the percentage, activation, and maturation of B cells in MRL/ lpr mice Serum levels of total IgG (A), IgG2a (B), and IgM (C) were determined by ELISA. (D) The percentage of B220 + B cells in spleens was analyzed by flow cytometry. (E and F)

Techniques Used: Activation Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

15) Product Images from "Human Vγ9Vδ2-T Cells Synergize CD4+ T Follicular Helper Cells to Produce Influenza Virus-Specific Antibody"

Article Title: Human Vγ9Vδ2-T Cells Synergize CD4+ T Follicular Helper Cells to Produce Influenza Virus-Specific Antibody

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00599

Vγ9Vδ2-T cells increased H9N2 virus-specific IgG and IgM productions in vivo . Specific immune cells-depleted and whole peripheral blood mononuclear cells (PBMC)-humanized mice were vaccinated with UV-inactivated H9N2 virus through i.p. pathway on day 0 and 7. Serum was collected on day 21 post the first vaccination. (A,B) Total IgG and IgM in serum (C,D) H9N2 virus-specific IgG and IgM in serum that treated with receptor destroyed enzyme (RDE) were determined by enzyme-linked immunosorbent assay. (E) H9N2-specific antibodies in serum that treated with RDE were determined by hemagglutination inhibition assay. The data shown were the represent of four independent experiments. (F) Representative histological analysis, immunohistological stainings for human CD4 T, B, and γδ-T cells in formalin-fixed paraffin-embedded spleen in humanized mice constructed with whole PBMCs after UV-H9N2 virus immunization. Scale bars = 100 μm. Bottom panels showed the higher-magnification views of the respective boxed areas in the top panels. Scale bars = 50 μm. The data shown are the mean ± SEM. * p
Figure Legend Snippet: Vγ9Vδ2-T cells increased H9N2 virus-specific IgG and IgM productions in vivo . Specific immune cells-depleted and whole peripheral blood mononuclear cells (PBMC)-humanized mice were vaccinated with UV-inactivated H9N2 virus through i.p. pathway on day 0 and 7. Serum was collected on day 21 post the first vaccination. (A,B) Total IgG and IgM in serum (C,D) H9N2 virus-specific IgG and IgM in serum that treated with receptor destroyed enzyme (RDE) were determined by enzyme-linked immunosorbent assay. (E) H9N2-specific antibodies in serum that treated with RDE were determined by hemagglutination inhibition assay. The data shown were the represent of four independent experiments. (F) Representative histological analysis, immunohistological stainings for human CD4 T, B, and γδ-T cells in formalin-fixed paraffin-embedded spleen in humanized mice constructed with whole PBMCs after UV-H9N2 virus immunization. Scale bars = 100 μm. Bottom panels showed the higher-magnification views of the respective boxed areas in the top panels. Scale bars = 50 μm. The data shown are the mean ± SEM. * p

Techniques Used: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, HI Assay, Formalin-fixed Paraffin-Embedded, Construct

Vγ9Vδ2-T cells facilitated influenza virus-specific antibody production. Naïve CD4 T cells were cultured with UV-H9N2 virus-pulsed dendritic cells for 4–5 days, then, the activated CD4 T cells were cultured with CD19 + B cells from same donor with or without Vγ9Vδ2-T cells for another 7–10 days. (A,B) Total IgG and IgM in supernatant on day 7 were detected by enzyme-linked immunosorbent assay (ELISA). (C,D) H9N2 virus-specific IgG and IgM were detected by ELISA on day 7. Each dot means one donor. The data shown are the mean ± SEM. * p
Figure Legend Snippet: Vγ9Vδ2-T cells facilitated influenza virus-specific antibody production. Naïve CD4 T cells were cultured with UV-H9N2 virus-pulsed dendritic cells for 4–5 days, then, the activated CD4 T cells were cultured with CD19 + B cells from same donor with or without Vγ9Vδ2-T cells for another 7–10 days. (A,B) Total IgG and IgM in supernatant on day 7 were detected by enzyme-linked immunosorbent assay (ELISA). (C,D) H9N2 virus-specific IgG and IgM were detected by ELISA on day 7. Each dot means one donor. The data shown are the mean ± SEM. * p

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

16) Product Images from "Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response"

Article Title: Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response

Journal: Pathogens

doi: 10.3390/pathogens6020016

In vitro immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on the Ig secretion by naive B cells induced by CpG ODNs. Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) CpG ODNs (1 µg/mL). After seven days of incubation, supernatants were collected and total IgM ( A ) and IgG ( B ) were quantified by ELISA. Cells stimulated with CpG ODNs alone (black bars) served as the positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of stimulation by CpG ODNs. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panel) experiments.
Figure Legend Snippet: In vitro immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on the Ig secretion by naive B cells induced by CpG ODNs. Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) CpG ODNs (1 µg/mL). After seven days of incubation, supernatants were collected and total IgM ( A ) and IgG ( B ) were quantified by ELISA. Cells stimulated with CpG ODNs alone (black bars) served as the positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of stimulation by CpG ODNs. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panel) experiments.

Techniques Used: In Vitro, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Positive Control

Titration of capsular polysaccharide (CPS)-specific antibodies in mice after infection with S. suis serotype 2, S. suis serotype 14, group B Streptococcus (GBS) serotype III or GBS serotype V. Mice were infected with 2 × 10 7 CFU of live S. suis serotype 2 strain P1/7 ( n = 60) ( A ); 5 × 10 6 CFU of live S. suis serotype 14 strain DAN13730 ( n = 40) ( B ); 2 × 10 6 CFU of live GBS serotype III strain COH-1 ( n = 40) ( C ); or 10 4 CFU of live GBS serotype V strain CJB111 ( n = 40) ( D ). Total Ig (IgG plus IgM) anti-CPS titers were determined by ELISA on Days 7 (in blue), 14 (in green) and 21 (in red) in surviving mice. IgM and IgG anti-CPS titers were determined on Day 21 (in red). For comparative purpose, total Ig (IgG plus IgM), IgM and IgG anti-protein (‘prot’) titers were also determined on Day 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. “C − ” represents a pool of control mice ( n = 3) injected with vehicle solution, whose titers were evaluated on Days 7, 14 and 21. * Statistically significant difference ( p
Figure Legend Snippet: Titration of capsular polysaccharide (CPS)-specific antibodies in mice after infection with S. suis serotype 2, S. suis serotype 14, group B Streptococcus (GBS) serotype III or GBS serotype V. Mice were infected with 2 × 10 7 CFU of live S. suis serotype 2 strain P1/7 ( n = 60) ( A ); 5 × 10 6 CFU of live S. suis serotype 14 strain DAN13730 ( n = 40) ( B ); 2 × 10 6 CFU of live GBS serotype III strain COH-1 ( n = 40) ( C ); or 10 4 CFU of live GBS serotype V strain CJB111 ( n = 40) ( D ). Total Ig (IgG plus IgM) anti-CPS titers were determined by ELISA on Days 7 (in blue), 14 (in green) and 21 (in red) in surviving mice. IgM and IgG anti-CPS titers were determined on Day 21 (in red). For comparative purpose, total Ig (IgG plus IgM), IgM and IgG anti-protein (‘prot’) titers were also determined on Day 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. “C − ” represents a pool of control mice ( n = 3) injected with vehicle solution, whose titers were evaluated on Days 7, 14 and 21. * Statistically significant difference ( p

Techniques Used: Titration, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Injection

Titration of CPS-specific antibodies in mice after immunization with purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS. Mice ( n = 8) were immunized with 2 µg of purified native or desialylated S. suis serotype 2 ( A ); S. suis serotype 14 ( B ); GBS serotype III ( C ); or GBS serotype V ( D ) CPS emulsified with STIMUNE ® . Total Ig (IgG plus IgM) anti-native CPS titers were determined by ELISA on Days 7, 14 and 21. “C − ” represents a pool of control mice ( n = 3) injected with STIMUNE ® only, whose titers were evaluated on Days 7, 14 and 21. Data from individual mice are presented, including the geometric mean with 95% confidence interval. * Statistically significant difference ( p
Figure Legend Snippet: Titration of CPS-specific antibodies in mice after immunization with purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS. Mice ( n = 8) were immunized with 2 µg of purified native or desialylated S. suis serotype 2 ( A ); S. suis serotype 14 ( B ); GBS serotype III ( C ); or GBS serotype V ( D ) CPS emulsified with STIMUNE ® . Total Ig (IgG plus IgM) anti-native CPS titers were determined by ELISA on Days 7, 14 and 21. “C − ” represents a pool of control mice ( n = 3) injected with STIMUNE ® only, whose titers were evaluated on Days 7, 14 and 21. Data from individual mice are presented, including the geometric mean with 95% confidence interval. * Statistically significant difference ( p

Techniques Used: Titration, Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay, Injection

Adjuvant effect of CpG oligodeoxynucleotides (ODNs) on the CPS-specific humoral response in mice immunized with purified native or desialylated S. suis serotype 2 CPS or purified S. pneumoniae serotype 3 CPS (PS3). ( A ) In a first set of experiments, mice ( n = 8) were immunized with 2 µg of purified native (n) or desialylated (dS) S. suis serotype 2 CPS (CPS S. suis ) or PS3 in PBS on Day 0 and 80 µg of CpG ODNs two days after. The control group ( n = 8) received CPS or PS3 on Day 0 and PBS two days later. The placebo group ( n = 3) received PBS or CpG ODNs only. Total Ig (IgG plus IgM) anti-native S. suis type 2 CPS or anti-PS3 titers were determined by ELISA on Days 7, 14 and 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. ( B ) In a second set of experiments, mice ( n = 5) were immunized as described in (A), but splenocytes were collected on Day 5 after immunization, and anti-CPS antibody-secreting cells (ASCs) were enumerated by ELISpot as described in the Materials and Methods section. Data are expressed as arithmetic means with SEM. ( C ) Visualization of PS3 (top) or native S. suis type 2 CPS (bottom) specific ASCs in ELISpot wells from splenocytes of mice obtained in (B). * p
Figure Legend Snippet: Adjuvant effect of CpG oligodeoxynucleotides (ODNs) on the CPS-specific humoral response in mice immunized with purified native or desialylated S. suis serotype 2 CPS or purified S. pneumoniae serotype 3 CPS (PS3). ( A ) In a first set of experiments, mice ( n = 8) were immunized with 2 µg of purified native (n) or desialylated (dS) S. suis serotype 2 CPS (CPS S. suis ) or PS3 in PBS on Day 0 and 80 µg of CpG ODNs two days after. The control group ( n = 8) received CPS or PS3 on Day 0 and PBS two days later. The placebo group ( n = 3) received PBS or CpG ODNs only. Total Ig (IgG plus IgM) anti-native S. suis type 2 CPS or anti-PS3 titers were determined by ELISA on Days 7, 14 and 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. ( B ) In a second set of experiments, mice ( n = 5) were immunized as described in (A), but splenocytes were collected on Day 5 after immunization, and anti-CPS antibody-secreting cells (ASCs) were enumerated by ELISpot as described in the Materials and Methods section. Data are expressed as arithmetic means with SEM. ( C ) Visualization of PS3 (top) or native S. suis type 2 CPS (bottom) specific ASCs in ELISpot wells from splenocytes of mice obtained in (B). * p

Techniques Used: Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

In vivo immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on ovalbumin (OVA)-specific antibody response. Mice ( n = 8) were co-immunized intraperitoneally with 10 µg of OVA and 2 µg of purified native S. suis serotype 2 ( A ), S. suis serotype 14 ( B ), GBS serotype III ( C ) or GBS serotype V ( D ) CPS in PBS on Days 0 and 21. Control group ( n = 8) received OVA only on Days 0 and 21. Total Ig (IgG plus IgM) anti-OVA titers were determined by ELISA on Days 7, 14, 21 and 35. “C − ” represents a pool of placebo mice ( n = 3) injected with PBS, whose titers were evaluated on Days 7, 14, 21 and 35. Data from individual mice are presented, including the geometric mean with 95% confidence interval. An arrow indicates secondary immunization. * p
Figure Legend Snippet: In vivo immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on ovalbumin (OVA)-specific antibody response. Mice ( n = 8) were co-immunized intraperitoneally with 10 µg of OVA and 2 µg of purified native S. suis serotype 2 ( A ), S. suis serotype 14 ( B ), GBS serotype III ( C ) or GBS serotype V ( D ) CPS in PBS on Days 0 and 21. Control group ( n = 8) received OVA only on Days 0 and 21. Total Ig (IgG plus IgM) anti-OVA titers were determined by ELISA on Days 7, 14, 21 and 35. “C − ” represents a pool of placebo mice ( n = 3) injected with PBS, whose titers were evaluated on Days 7, 14, 21 and 35. Data from individual mice are presented, including the geometric mean with 95% confidence interval. An arrow indicates secondary immunization. * p

Techniques Used: In Vivo, Purification, Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection

In vitro immunomodulatory effect of purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on BAFF/IL-4-induced Ig secretion by naive B cells. ( A , B ) Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) BAFF (1 µg/mL) along with IL-4 (50 ng/mL); ( C , D ) in order to evaluate the influence of sialic acid, cells were also incubated with desialylated (dS) S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS in parallel to cells incubated with the respective native CPS (n) as described in ( A , B ). After seven days of incubation, supernatants were collected, and total IgM ( A , C ) and IgG ( B , D ) was quantified by ELISA. Cells stimulated with BAFF/IL-4 alone (black bars) served as a positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of BAFF/IL-4 stimulation. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panels) experiments. * p
Figure Legend Snippet: In vitro immunomodulatory effect of purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on BAFF/IL-4-induced Ig secretion by naive B cells. ( A , B ) Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) BAFF (1 µg/mL) along with IL-4 (50 ng/mL); ( C , D ) in order to evaluate the influence of sialic acid, cells were also incubated with desialylated (dS) S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS in parallel to cells incubated with the respective native CPS (n) as described in ( A , B ). After seven days of incubation, supernatants were collected, and total IgM ( A , C ) and IgG ( B , D ) was quantified by ELISA. Cells stimulated with BAFF/IL-4 alone (black bars) served as a positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of BAFF/IL-4 stimulation. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panels) experiments. * p

Techniques Used: In Vitro, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Positive Control

17) Product Images from "Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals"

Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01364

Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P
Figure Legend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P

Techniques Used: Activity Assay, Indirect Immunoperoxidase Assay, Software, Activation Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Mouse Assay

TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P
Figure Legend Snippet: TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P

Techniques Used: Labeling, Enzyme-linked Immunosorbent Assay, Cell Culture, Mouse Assay, Plasmid Preparation

Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P
Figure Legend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P

Techniques Used: Mouse Assay, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P
Figure Legend Snippet: TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P

Techniques Used: Activation Assay, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Expressing, Mouse Assay, Plasmid Preparation, Injection

18) Product Images from "Systemic and mucosal humoral immune responses induced by the JY-adjuvanted nasal spray H7N9 vaccine in mice"

Article Title: Systemic and mucosal humoral immune responses induced by the JY-adjuvanted nasal spray H7N9 vaccine in mice

Journal: Emerging Microbes & Infections

doi: 10.1038/s41426-018-0133-y

Comparison of the adjuvant effect of different adjuvants on the nasal spray H7N9 vaccine. Mice were intranasally immunized with 4.5 μg HA twice (3-week interval) and with different adjuvants. Serum and BALF were collected at day 21 after the last immunization, and the titers of HI and IgG in serum and of sIgA in BALF were detected. The data are shown as the geometric mean of mice in each group with the corresponding SD on a log 2 scale, and the results were compared using Student’s t -test. Differences with a P -value
Figure Legend Snippet: Comparison of the adjuvant effect of different adjuvants on the nasal spray H7N9 vaccine. Mice were intranasally immunized with 4.5 μg HA twice (3-week interval) and with different adjuvants. Serum and BALF were collected at day 21 after the last immunization, and the titers of HI and IgG in serum and of sIgA in BALF were detected. The data are shown as the geometric mean of mice in each group with the corresponding SD on a log 2 scale, and the results were compared using Student’s t -test. Differences with a P -value

Techniques Used: Mouse Assay

Optimal dose selection. BALB/c mice were randomly divided into five groups and intranasally immunized with the JY-adjuvanted nasal spray H7N9 vaccine with a 21-day interval but with different HA contents: 15, 9, 4.5, 2.25, or 1.13 μg. An NS control group was also included. Serum and BALF were collected 21 days after the last immunization. The titers of HI and anti-HA IgG in serum and of sIgA in BALF were detected using the HI assay or ELISA. The data are shown as the geometric mean of all mice in each group with the corresponding SD on a log 2 scale, and the results were compared using Student’s t -test. Differences with a P -value
Figure Legend Snippet: Optimal dose selection. BALB/c mice were randomly divided into five groups and intranasally immunized with the JY-adjuvanted nasal spray H7N9 vaccine with a 21-day interval but with different HA contents: 15, 9, 4.5, 2.25, or 1.13 μg. An NS control group was also included. Serum and BALF were collected 21 days after the last immunization. The titers of HI and anti-HA IgG in serum and of sIgA in BALF were detected using the HI assay or ELISA. The data are shown as the geometric mean of all mice in each group with the corresponding SD on a log 2 scale, and the results were compared using Student’s t -test. Differences with a P -value

Techniques Used: Selection, Mouse Assay, HI Assay, Enzyme-linked Immunosorbent Assay

Optimal immunization interval. BALB/c mice were randomly divided into four groups and intranasally immunized with the JY-adjuvanted H7N9 nasal spray vaccine containing 4.5 μg HA at 7-day, 14-day, and 21-day intervals or an NS control. Three weeks after the last immunization, serum and BALF were collected. The titers of HI and anti-HA IgG in serum and of sIgA in BALF were detected using the HI assay or ELISA. The data are shown as the geometric mean of all mice in each group with the corresponding SD on a log 2 scale, and the results were compared using Student’s t -test. Differences with a P -value
Figure Legend Snippet: Optimal immunization interval. BALB/c mice were randomly divided into four groups and intranasally immunized with the JY-adjuvanted H7N9 nasal spray vaccine containing 4.5 μg HA at 7-day, 14-day, and 21-day intervals or an NS control. Three weeks after the last immunization, serum and BALF were collected. The titers of HI and anti-HA IgG in serum and of sIgA in BALF were detected using the HI assay or ELISA. The data are shown as the geometric mean of all mice in each group with the corresponding SD on a log 2 scale, and the results were compared using Student’s t -test. Differences with a P -value

Techniques Used: Mouse Assay, HI Assay, Enzyme-linked Immunosorbent Assay

Effect of JY adjuvant on the immunogenicity of the nasal spray H7N9 or H1N1 vaccine. Mice were immunized twice (1-week interval) with the H7N9 or H1N1 influenza vaccine with or without JY adjuvant. The HI titers in serum were determined by the HI assay with virus H7N9 (A/shanghai/02/2013) or H1N1 (A/California/07/2009). The subclasses of the anti-HA IgG (IgG2a and IgG1) in serum were detected by ELISA. The data are shown as the geometric mean of mice in each group with their corresponding SD on a log 2 scale, and the results were compared using Student’s t -test. Differences with a P -value
Figure Legend Snippet: Effect of JY adjuvant on the immunogenicity of the nasal spray H7N9 or H1N1 vaccine. Mice were immunized twice (1-week interval) with the H7N9 or H1N1 influenza vaccine with or without JY adjuvant. The HI titers in serum were determined by the HI assay with virus H7N9 (A/shanghai/02/2013) or H1N1 (A/California/07/2009). The subclasses of the anti-HA IgG (IgG2a and IgG1) in serum were detected by ELISA. The data are shown as the geometric mean of mice in each group with their corresponding SD on a log 2 scale, and the results were compared using Student’s t -test. Differences with a P -value

Techniques Used: Mouse Assay, HI Assay, Enzyme-linked Immunosorbent Assay

Immunogenicity comparison of the nasal spray H7N9 vaccine and the intramuscular vaccine. Mice were intranasally or intramuscularly immunized with vaccines once or twice (3-week interval). Serum and BALF were collected 21 days after the last immunization, and then the titers of HI and anti-HA IgG in serum and of sIgA in BALF were determined. The data are shown as the geometric mean of all mice in each group with the corresponding SD on a log 2 scale, and the results were compared using Student’s t -test. Differences with a P -value
Figure Legend Snippet: Immunogenicity comparison of the nasal spray H7N9 vaccine and the intramuscular vaccine. Mice were intranasally or intramuscularly immunized with vaccines once or twice (3-week interval). Serum and BALF were collected 21 days after the last immunization, and then the titers of HI and anti-HA IgG in serum and of sIgA in BALF were determined. The data are shown as the geometric mean of all mice in each group with the corresponding SD on a log 2 scale, and the results were compared using Student’s t -test. Differences with a P -value

Techniques Used: Mouse Assay

19) Product Images from "Dichotomy in FcγRIIB deficiency and autoimmune-prone SLAM haplotype clarifies the roles of the Fc receptor in development of autoantibodies and glomerulonephritis"

Article Title: Dichotomy in FcγRIIB deficiency and autoimmune-prone SLAM haplotype clarifies the roles of the Fc receptor in development of autoantibodies and glomerulonephritis

Journal: BMC Immunology

doi: 10.1186/s12865-014-0047-y

Female-biased production of anti-nuclear antibodies in RIIB −/− mice. (A) The total IgM and IgG levels in sera from different lines, including B6, RIIB −/− , SLAM 129 , and RIIB −/− SLAM 129 , at 24–28 weeks of age of both genders were measured by ELISA. For IgM and IgG determinations in male B6 mice, n =3 and 5, respectively; for other determinations, n ≥8. The data in each panel are from two separate measurements and are presented as means ± S.D. * P
Figure Legend Snippet: Female-biased production of anti-nuclear antibodies in RIIB −/− mice. (A) The total IgM and IgG levels in sera from different lines, including B6, RIIB −/− , SLAM 129 , and RIIB −/− SLAM 129 , at 24–28 weeks of age of both genders were measured by ELISA. For IgM and IgG determinations in male B6 mice, n =3 and 5, respectively; for other determinations, n ≥8. The data in each panel are from two separate measurements and are presented as means ± S.D. * P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

20) Product Images from "CD180 Ligation Inhibits TLR7- and TLR9-Mediated Activation of Macrophages and Dendritic Cells Through the Lyn-SHP-1/2 Axis in Murine Lupus"

Article Title: CD180 Ligation Inhibits TLR7- and TLR9-Mediated Activation of Macrophages and Dendritic Cells Through the Lyn-SHP-1/2 Axis in Murine Lupus

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02643

Treatment effect of anti-CD180 Ab on lupus-symptoms of MRL/ lpr mice. (A) Proteinuria was collected weekly and determined by ELISA. (B) Representative images of marked splenomegaly in all groups of mice. (C,D) ELISA analysis of the anti-dsDNA antibody (C) and anti-RNA antibody (D) in serum. (E) H E staining of the kidney sections from all groups of mice. (F) Immunofluorescence staining to detect IgG deposit in the kidney sections. (G) Immunofluorescence staining to detect IgM deposit in the kidney sections. (H–J) Flow cytometric analysis of CD86 expression on peritoneal macrophages (H) , splenic macrophages (I) , and DCs (J) in all groups of mice. The data are shown as the means ± SEM ( n = 6 mice/group). ** p
Figure Legend Snippet: Treatment effect of anti-CD180 Ab on lupus-symptoms of MRL/ lpr mice. (A) Proteinuria was collected weekly and determined by ELISA. (B) Representative images of marked splenomegaly in all groups of mice. (C,D) ELISA analysis of the anti-dsDNA antibody (C) and anti-RNA antibody (D) in serum. (E) H E staining of the kidney sections from all groups of mice. (F) Immunofluorescence staining to detect IgG deposit in the kidney sections. (G) Immunofluorescence staining to detect IgM deposit in the kidney sections. (H–J) Flow cytometric analysis of CD86 expression on peritoneal macrophages (H) , splenic macrophages (I) , and DCs (J) in all groups of mice. The data are shown as the means ± SEM ( n = 6 mice/group). ** p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Flow Cytometry, Expressing

Treatment effect of anti-CD180 Ab on lupus-symptoms of IMQ-treated mice. (A) Treatment schematic of anti-CD180 Ab and IgG2b injection to mice following epicutaneous application of the TLR7 agonist imiquimod. (B) Proteinuria was collected weekly and determined by ELISA. (C) Representative images of marked splenomegaly in all groups of mice. (D,E) ELISA analysis of the anti-dsDNA antibody (D) and anti-RNA antibody (E) in serum. (F) H E staining of the kidney sections from all groups of mice. (G) Immunofluorescence staining to detect IgG deposit in the kidney sections. (H) Immunofluorescence staining to detect IgM deposit in the kidney sections. (I–K) Flow cytometric analysis of CD86 expression on peritoneal macrophages (I) , splenic macrophages (J) , and DCs (K) in all groups of mice. The data are shown as the means ± SEM ( n = 7 mice/group). * p
Figure Legend Snippet: Treatment effect of anti-CD180 Ab on lupus-symptoms of IMQ-treated mice. (A) Treatment schematic of anti-CD180 Ab and IgG2b injection to mice following epicutaneous application of the TLR7 agonist imiquimod. (B) Proteinuria was collected weekly and determined by ELISA. (C) Representative images of marked splenomegaly in all groups of mice. (D,E) ELISA analysis of the anti-dsDNA antibody (D) and anti-RNA antibody (E) in serum. (F) H E staining of the kidney sections from all groups of mice. (G) Immunofluorescence staining to detect IgG deposit in the kidney sections. (H) Immunofluorescence staining to detect IgM deposit in the kidney sections. (I–K) Flow cytometric analysis of CD86 expression on peritoneal macrophages (I) , splenic macrophages (J) , and DCs (K) in all groups of mice. The data are shown as the means ± SEM ( n = 7 mice/group). * p

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Flow Cytometry, Expressing

21) Product Images from "Identification of Liver Epithelial Cell-derived Ig Expression in μ chain-deficient mice"

Article Title: Identification of Liver Epithelial Cell-derived Ig Expression in μ chain-deficient mice

Journal: Scientific Reports

doi: 10.1038/srep23669

IgM, IgG, IgD, IgA, Ig κ and Igλ detection in serum and liver cells. ( A ) Ig expression level in serum and liver lysate of WT mice and μMT mice were measured by Mouse Ig Isotyping Array. WT serum was diluted 100 times, while serum from μMT mice was diluted 50 times. The statistical analysis was performed under the diluted condition of serum and the dilution difference was not taken into account. Igs were detected in 30 μg liver protein from WT mice or μMT mice. ( B ) The concentrations of IgM in serum and liver lysate from WT mice and μMT mice were detected by ELISA. ***P
Figure Legend Snippet: IgM, IgG, IgD, IgA, Ig κ and Igλ detection in serum and liver cells. ( A ) Ig expression level in serum and liver lysate of WT mice and μMT mice were measured by Mouse Ig Isotyping Array. WT serum was diluted 100 times, while serum from μMT mice was diluted 50 times. The statistical analysis was performed under the diluted condition of serum and the dilution difference was not taken into account. Igs were detected in 30 μg liver protein from WT mice or μMT mice. ( B ) The concentrations of IgM in serum and liver lysate from WT mice and μMT mice were detected by ELISA. ***P

Techniques Used: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay

Identification of B cells and Igs in μMT mice. ( A ) B lymphocyte and T lymphocyte in peripheral blood of WT mice and μMT mice were detected with PE anti-mouse B220 and FITC anti-mouse CD3 by FACS. ( B ) Pro-B cell (CD43 + B220 low ) and pre-B cell (CD43 − B220 + ) in BM of WT mice and μMT mice were detected with PE anti-mouse CD43 and PE/Cy7 anti-mouse B220 by FACS. The cells in the upper left circle were pre-B cells, and the cells in the bottom right circle were pro-B cells. ( C ) Mature B cell (B220 + IgM + ) in BM of WT mice and μMT mice were detected with FITC anti-mouse IgM and PE/Cy7 anti-mouse B220 by FACS. The circle showed mature B cells. ( D ) The concentrations of IgM and IgG in serum from WT mice and μMT mice were detected by ELISA. ***P
Figure Legend Snippet: Identification of B cells and Igs in μMT mice. ( A ) B lymphocyte and T lymphocyte in peripheral blood of WT mice and μMT mice were detected with PE anti-mouse B220 and FITC anti-mouse CD3 by FACS. ( B ) Pro-B cell (CD43 + B220 low ) and pre-B cell (CD43 − B220 + ) in BM of WT mice and μMT mice were detected with PE anti-mouse CD43 and PE/Cy7 anti-mouse B220 by FACS. The cells in the upper left circle were pre-B cells, and the cells in the bottom right circle were pro-B cells. ( C ) Mature B cell (B220 + IgM + ) in BM of WT mice and μMT mice were detected with FITC anti-mouse IgM and PE/Cy7 anti-mouse B220 by FACS. The circle showed mature B cells. ( D ) The concentrations of IgM and IgG in serum from WT mice and μMT mice were detected by ELISA. ***P

Techniques Used: Mouse Assay, FACS, Enzyme-linked Immunosorbent Assay

22) Product Images from "Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response"

Article Title: Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response

Journal: Pathogens

doi: 10.3390/pathogens6020016

In vitro immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on the Ig secretion by naive B cells induced by CpG ODNs. Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) CpG ODNs (1 µg/mL). After seven days of incubation, supernatants were collected and total IgM ( A ) and IgG ( B ) were quantified by ELISA. Cells stimulated with CpG ODNs alone (black bars) served as the positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of stimulation by CpG ODNs. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panel) experiments.
Figure Legend Snippet: In vitro immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on the Ig secretion by naive B cells induced by CpG ODNs. Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) CpG ODNs (1 µg/mL). After seven days of incubation, supernatants were collected and total IgM ( A ) and IgG ( B ) were quantified by ELISA. Cells stimulated with CpG ODNs alone (black bars) served as the positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of stimulation by CpG ODNs. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panel) experiments.

Techniques Used: In Vitro, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Positive Control

Titration of capsular polysaccharide (CPS)-specific antibodies in mice after infection with S. suis serotype 2, S. suis serotype 14, group B Streptococcus (GBS) serotype III or GBS serotype V. Mice were infected with 2 × 10 7 CFU of live S. suis serotype 2 strain P1/7 ( n = 60) ( A ); 5 × 10 6 CFU of live S. suis serotype 14 strain DAN13730 ( n = 40) ( B ); 2 × 10 6 CFU of live GBS serotype III strain COH-1 ( n = 40) ( C ); or 10 4 CFU of live GBS serotype V strain CJB111 ( n = 40) ( D ). Total Ig (IgG plus IgM) anti-CPS titers were determined by ELISA on Days 7 (in blue), 14 (in green) and 21 (in red) in surviving mice. IgM and IgG anti-CPS titers were determined on Day 21 (in red). For comparative purpose, total Ig (IgG plus IgM), IgM and IgG anti-protein (‘prot’) titers were also determined on Day 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. “C − ” represents a pool of control mice ( n = 3) injected with vehicle solution, whose titers were evaluated on Days 7, 14 and 21. * Statistically significant difference ( p
Figure Legend Snippet: Titration of capsular polysaccharide (CPS)-specific antibodies in mice after infection with S. suis serotype 2, S. suis serotype 14, group B Streptococcus (GBS) serotype III or GBS serotype V. Mice were infected with 2 × 10 7 CFU of live S. suis serotype 2 strain P1/7 ( n = 60) ( A ); 5 × 10 6 CFU of live S. suis serotype 14 strain DAN13730 ( n = 40) ( B ); 2 × 10 6 CFU of live GBS serotype III strain COH-1 ( n = 40) ( C ); or 10 4 CFU of live GBS serotype V strain CJB111 ( n = 40) ( D ). Total Ig (IgG plus IgM) anti-CPS titers were determined by ELISA on Days 7 (in blue), 14 (in green) and 21 (in red) in surviving mice. IgM and IgG anti-CPS titers were determined on Day 21 (in red). For comparative purpose, total Ig (IgG plus IgM), IgM and IgG anti-protein (‘prot’) titers were also determined on Day 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. “C − ” represents a pool of control mice ( n = 3) injected with vehicle solution, whose titers were evaluated on Days 7, 14 and 21. * Statistically significant difference ( p

Techniques Used: Titration, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Injection

Titration of CPS-specific antibodies in mice after immunization with purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS. Mice ( n = 8) were immunized with 2 µg of purified native or desialylated S. suis serotype 2 ( A ); S. suis serotype 14 ( B ); GBS serotype III ( C ); or GBS serotype V ( D ) CPS emulsified with STIMUNE ® . Total Ig (IgG plus IgM) anti-native CPS titers were determined by ELISA on Days 7, 14 and 21. “C − ” represents a pool of control mice ( n = 3) injected with STIMUNE ® only, whose titers were evaluated on Days 7, 14 and 21. Data from individual mice are presented, including the geometric mean with 95% confidence interval. * Statistically significant difference ( p
Figure Legend Snippet: Titration of CPS-specific antibodies in mice after immunization with purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS. Mice ( n = 8) were immunized with 2 µg of purified native or desialylated S. suis serotype 2 ( A ); S. suis serotype 14 ( B ); GBS serotype III ( C ); or GBS serotype V ( D ) CPS emulsified with STIMUNE ® . Total Ig (IgG plus IgM) anti-native CPS titers were determined by ELISA on Days 7, 14 and 21. “C − ” represents a pool of control mice ( n = 3) injected with STIMUNE ® only, whose titers were evaluated on Days 7, 14 and 21. Data from individual mice are presented, including the geometric mean with 95% confidence interval. * Statistically significant difference ( p

Techniques Used: Titration, Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay, Injection

Adjuvant effect of CpG oligodeoxynucleotides (ODNs) on the CPS-specific humoral response in mice immunized with purified native or desialylated S. suis serotype 2 CPS or purified S. pneumoniae serotype 3 CPS (PS3). ( A ) In a first set of experiments, mice ( n = 8) were immunized with 2 µg of purified native (n) or desialylated (dS) S. suis serotype 2 CPS (CPS S. suis ) or PS3 in PBS on Day 0 and 80 µg of CpG ODNs two days after. The control group ( n = 8) received CPS or PS3 on Day 0 and PBS two days later. The placebo group ( n = 3) received PBS or CpG ODNs only. Total Ig (IgG plus IgM) anti-native S. suis type 2 CPS or anti-PS3 titers were determined by ELISA on Days 7, 14 and 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. ( B ) In a second set of experiments, mice ( n = 5) were immunized as described in (A), but splenocytes were collected on Day 5 after immunization, and anti-CPS antibody-secreting cells (ASCs) were enumerated by ELISpot as described in the Materials and Methods section. Data are expressed as arithmetic means with SEM. ( C ) Visualization of PS3 (top) or native S. suis type 2 CPS (bottom) specific ASCs in ELISpot wells from splenocytes of mice obtained in (B). * p
Figure Legend Snippet: Adjuvant effect of CpG oligodeoxynucleotides (ODNs) on the CPS-specific humoral response in mice immunized with purified native or desialylated S. suis serotype 2 CPS or purified S. pneumoniae serotype 3 CPS (PS3). ( A ) In a first set of experiments, mice ( n = 8) were immunized with 2 µg of purified native (n) or desialylated (dS) S. suis serotype 2 CPS (CPS S. suis ) or PS3 in PBS on Day 0 and 80 µg of CpG ODNs two days after. The control group ( n = 8) received CPS or PS3 on Day 0 and PBS two days later. The placebo group ( n = 3) received PBS or CpG ODNs only. Total Ig (IgG plus IgM) anti-native S. suis type 2 CPS or anti-PS3 titers were determined by ELISA on Days 7, 14 and 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. ( B ) In a second set of experiments, mice ( n = 5) were immunized as described in (A), but splenocytes were collected on Day 5 after immunization, and anti-CPS antibody-secreting cells (ASCs) were enumerated by ELISpot as described in the Materials and Methods section. Data are expressed as arithmetic means with SEM. ( C ) Visualization of PS3 (top) or native S. suis type 2 CPS (bottom) specific ASCs in ELISpot wells from splenocytes of mice obtained in (B). * p

Techniques Used: Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

In vivo immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on ovalbumin (OVA)-specific antibody response. Mice ( n = 8) were co-immunized intraperitoneally with 10 µg of OVA and 2 µg of purified native S. suis serotype 2 ( A ), S. suis serotype 14 ( B ), GBS serotype III ( C ) or GBS serotype V ( D ) CPS in PBS on Days 0 and 21. Control group ( n = 8) received OVA only on Days 0 and 21. Total Ig (IgG plus IgM) anti-OVA titers were determined by ELISA on Days 7, 14, 21 and 35. “C − ” represents a pool of placebo mice ( n = 3) injected with PBS, whose titers were evaluated on Days 7, 14, 21 and 35. Data from individual mice are presented, including the geometric mean with 95% confidence interval. An arrow indicates secondary immunization. * p
Figure Legend Snippet: In vivo immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on ovalbumin (OVA)-specific antibody response. Mice ( n = 8) were co-immunized intraperitoneally with 10 µg of OVA and 2 µg of purified native S. suis serotype 2 ( A ), S. suis serotype 14 ( B ), GBS serotype III ( C ) or GBS serotype V ( D ) CPS in PBS on Days 0 and 21. Control group ( n = 8) received OVA only on Days 0 and 21. Total Ig (IgG plus IgM) anti-OVA titers were determined by ELISA on Days 7, 14, 21 and 35. “C − ” represents a pool of placebo mice ( n = 3) injected with PBS, whose titers were evaluated on Days 7, 14, 21 and 35. Data from individual mice are presented, including the geometric mean with 95% confidence interval. An arrow indicates secondary immunization. * p

Techniques Used: In Vivo, Purification, Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection

In vitro immunomodulatory effect of purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on BAFF/IL-4-induced Ig secretion by naive B cells. ( A , B ) Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) BAFF (1 µg/mL) along with IL-4 (50 ng/mL); ( C , D ) in order to evaluate the influence of sialic acid, cells were also incubated with desialylated (dS) S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS in parallel to cells incubated with the respective native CPS (n) as described in ( A , B ). After seven days of incubation, supernatants were collected, and total IgM ( A , C ) and IgG ( B , D ) was quantified by ELISA. Cells stimulated with BAFF/IL-4 alone (black bars) served as a positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of BAFF/IL-4 stimulation. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panels) experiments. * p
Figure Legend Snippet: In vitro immunomodulatory effect of purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on BAFF/IL-4-induced Ig secretion by naive B cells. ( A , B ) Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) BAFF (1 µg/mL) along with IL-4 (50 ng/mL); ( C , D ) in order to evaluate the influence of sialic acid, cells were also incubated with desialylated (dS) S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS in parallel to cells incubated with the respective native CPS (n) as described in ( A , B ). After seven days of incubation, supernatants were collected, and total IgM ( A , C ) and IgG ( B , D ) was quantified by ELISA. Cells stimulated with BAFF/IL-4 alone (black bars) served as a positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of BAFF/IL-4 stimulation. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panels) experiments. * p

Techniques Used: In Vitro, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Positive Control

23) Product Images from "Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response"

Article Title: Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response

Journal: Pathogens

doi: 10.3390/pathogens6020016

In vitro immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on the Ig secretion by naive B cells induced by CpG ODNs. Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) CpG ODNs (1 µg/mL). After seven days of incubation, supernatants were collected and total IgM ( A ) and IgG ( B ) were quantified by ELISA. Cells stimulated with CpG ODNs alone (black bars) served as the positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of stimulation by CpG ODNs. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panel) experiments.
Figure Legend Snippet: In vitro immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on the Ig secretion by naive B cells induced by CpG ODNs. Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) CpG ODNs (1 µg/mL). After seven days of incubation, supernatants were collected and total IgM ( A ) and IgG ( B ) were quantified by ELISA. Cells stimulated with CpG ODNs alone (black bars) served as the positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of stimulation by CpG ODNs. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panel) experiments.

Techniques Used: In Vitro, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Positive Control

Titration of capsular polysaccharide (CPS)-specific antibodies in mice after infection with S. suis serotype 2, S. suis serotype 14, group B Streptococcus (GBS) serotype III or GBS serotype V. Mice were infected with 2 × 10 7 CFU of live S. suis serotype 2 strain P1/7 ( n = 60) ( A ); 5 × 10 6 CFU of live S. suis serotype 14 strain DAN13730 ( n = 40) ( B ); 2 × 10 6 CFU of live GBS serotype III strain COH-1 ( n = 40) ( C ); or 10 4 CFU of live GBS serotype V strain CJB111 ( n = 40) ( D ). Total Ig (IgG plus IgM) anti-CPS titers were determined by ELISA on Days 7 (in blue), 14 (in green) and 21 (in red) in surviving mice. IgM and IgG anti-CPS titers were determined on Day 21 (in red). For comparative purpose, total Ig (IgG plus IgM), IgM and IgG anti-protein (‘prot’) titers were also determined on Day 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. “C − ” represents a pool of control mice ( n = 3) injected with vehicle solution, whose titers were evaluated on Days 7, 14 and 21. * Statistically significant difference ( p
Figure Legend Snippet: Titration of capsular polysaccharide (CPS)-specific antibodies in mice after infection with S. suis serotype 2, S. suis serotype 14, group B Streptococcus (GBS) serotype III or GBS serotype V. Mice were infected with 2 × 10 7 CFU of live S. suis serotype 2 strain P1/7 ( n = 60) ( A ); 5 × 10 6 CFU of live S. suis serotype 14 strain DAN13730 ( n = 40) ( B ); 2 × 10 6 CFU of live GBS serotype III strain COH-1 ( n = 40) ( C ); or 10 4 CFU of live GBS serotype V strain CJB111 ( n = 40) ( D ). Total Ig (IgG plus IgM) anti-CPS titers were determined by ELISA on Days 7 (in blue), 14 (in green) and 21 (in red) in surviving mice. IgM and IgG anti-CPS titers were determined on Day 21 (in red). For comparative purpose, total Ig (IgG plus IgM), IgM and IgG anti-protein (‘prot’) titers were also determined on Day 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. “C − ” represents a pool of control mice ( n = 3) injected with vehicle solution, whose titers were evaluated on Days 7, 14 and 21. * Statistically significant difference ( p

Techniques Used: Titration, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Injection

Titration of CPS-specific antibodies in mice after immunization with purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS. Mice ( n = 8) were immunized with 2 µg of purified native or desialylated S. suis serotype 2 ( A ); S. suis serotype 14 ( B ); GBS serotype III ( C ); or GBS serotype V ( D ) CPS emulsified with STIMUNE ® . Total Ig (IgG plus IgM) anti-native CPS titers were determined by ELISA on Days 7, 14 and 21. “C − ” represents a pool of control mice ( n = 3) injected with STIMUNE ® only, whose titers were evaluated on Days 7, 14 and 21. Data from individual mice are presented, including the geometric mean with 95% confidence interval. * Statistically significant difference ( p
Figure Legend Snippet: Titration of CPS-specific antibodies in mice after immunization with purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS. Mice ( n = 8) were immunized with 2 µg of purified native or desialylated S. suis serotype 2 ( A ); S. suis serotype 14 ( B ); GBS serotype III ( C ); or GBS serotype V ( D ) CPS emulsified with STIMUNE ® . Total Ig (IgG plus IgM) anti-native CPS titers were determined by ELISA on Days 7, 14 and 21. “C − ” represents a pool of control mice ( n = 3) injected with STIMUNE ® only, whose titers were evaluated on Days 7, 14 and 21. Data from individual mice are presented, including the geometric mean with 95% confidence interval. * Statistically significant difference ( p

Techniques Used: Titration, Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay, Injection

Adjuvant effect of CpG oligodeoxynucleotides (ODNs) on the CPS-specific humoral response in mice immunized with purified native or desialylated S. suis serotype 2 CPS or purified S. pneumoniae serotype 3 CPS (PS3). ( A ) In a first set of experiments, mice ( n = 8) were immunized with 2 µg of purified native (n) or desialylated (dS) S. suis serotype 2 CPS (CPS S. suis ) or PS3 in PBS on Day 0 and 80 µg of CpG ODNs two days after. The control group ( n = 8) received CPS or PS3 on Day 0 and PBS two days later. The placebo group ( n = 3) received PBS or CpG ODNs only. Total Ig (IgG plus IgM) anti-native S. suis type 2 CPS or anti-PS3 titers were determined by ELISA on Days 7, 14 and 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. ( B ) In a second set of experiments, mice ( n = 5) were immunized as described in (A), but splenocytes were collected on Day 5 after immunization, and anti-CPS antibody-secreting cells (ASCs) were enumerated by ELISpot as described in the Materials and Methods section. Data are expressed as arithmetic means with SEM. ( C ) Visualization of PS3 (top) or native S. suis type 2 CPS (bottom) specific ASCs in ELISpot wells from splenocytes of mice obtained in (B). * p
Figure Legend Snippet: Adjuvant effect of CpG oligodeoxynucleotides (ODNs) on the CPS-specific humoral response in mice immunized with purified native or desialylated S. suis serotype 2 CPS or purified S. pneumoniae serotype 3 CPS (PS3). ( A ) In a first set of experiments, mice ( n = 8) were immunized with 2 µg of purified native (n) or desialylated (dS) S. suis serotype 2 CPS (CPS S. suis ) or PS3 in PBS on Day 0 and 80 µg of CpG ODNs two days after. The control group ( n = 8) received CPS or PS3 on Day 0 and PBS two days later. The placebo group ( n = 3) received PBS or CpG ODNs only. Total Ig (IgG plus IgM) anti-native S. suis type 2 CPS or anti-PS3 titers were determined by ELISA on Days 7, 14 and 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. ( B ) In a second set of experiments, mice ( n = 5) were immunized as described in (A), but splenocytes were collected on Day 5 after immunization, and anti-CPS antibody-secreting cells (ASCs) were enumerated by ELISpot as described in the Materials and Methods section. Data are expressed as arithmetic means with SEM. ( C ) Visualization of PS3 (top) or native S. suis type 2 CPS (bottom) specific ASCs in ELISpot wells from splenocytes of mice obtained in (B). * p

Techniques Used: Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

In vivo immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on ovalbumin (OVA)-specific antibody response. Mice ( n = 8) were co-immunized intraperitoneally with 10 µg of OVA and 2 µg of purified native S. suis serotype 2 ( A ), S. suis serotype 14 ( B ), GBS serotype III ( C ) or GBS serotype V ( D ) CPS in PBS on Days 0 and 21. Control group ( n = 8) received OVA only on Days 0 and 21. Total Ig (IgG plus IgM) anti-OVA titers were determined by ELISA on Days 7, 14, 21 and 35. “C − ” represents a pool of placebo mice ( n = 3) injected with PBS, whose titers were evaluated on Days 7, 14, 21 and 35. Data from individual mice are presented, including the geometric mean with 95% confidence interval. An arrow indicates secondary immunization. * p
Figure Legend Snippet: In vivo immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on ovalbumin (OVA)-specific antibody response. Mice ( n = 8) were co-immunized intraperitoneally with 10 µg of OVA and 2 µg of purified native S. suis serotype 2 ( A ), S. suis serotype 14 ( B ), GBS serotype III ( C ) or GBS serotype V ( D ) CPS in PBS on Days 0 and 21. Control group ( n = 8) received OVA only on Days 0 and 21. Total Ig (IgG plus IgM) anti-OVA titers were determined by ELISA on Days 7, 14, 21 and 35. “C − ” represents a pool of placebo mice ( n = 3) injected with PBS, whose titers were evaluated on Days 7, 14, 21 and 35. Data from individual mice are presented, including the geometric mean with 95% confidence interval. An arrow indicates secondary immunization. * p

Techniques Used: In Vivo, Purification, Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection

In vitro immunomodulatory effect of purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on BAFF/IL-4-induced Ig secretion by naive B cells. ( A , B ) Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) BAFF (1 µg/mL) along with IL-4 (50 ng/mL); ( C , D ) in order to evaluate the influence of sialic acid, cells were also incubated with desialylated (dS) S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS in parallel to cells incubated with the respective native CPS (n) as described in ( A , B ). After seven days of incubation, supernatants were collected, and total IgM ( A , C ) and IgG ( B , D ) was quantified by ELISA. Cells stimulated with BAFF/IL-4 alone (black bars) served as a positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of BAFF/IL-4 stimulation. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panels) experiments. * p
Figure Legend Snippet: In vitro immunomodulatory effect of purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on BAFF/IL-4-induced Ig secretion by naive B cells. ( A , B ) Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) BAFF (1 µg/mL) along with IL-4 (50 ng/mL); ( C , D ) in order to evaluate the influence of sialic acid, cells were also incubated with desialylated (dS) S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS in parallel to cells incubated with the respective native CPS (n) as described in ( A , B ). After seven days of incubation, supernatants were collected, and total IgM ( A , C ) and IgG ( B , D ) was quantified by ELISA. Cells stimulated with BAFF/IL-4 alone (black bars) served as a positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of BAFF/IL-4 stimulation. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panels) experiments. * p

Techniques Used: In Vitro, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Positive Control

24) Product Images from "Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals"

Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01364

Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P
Figure Legend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P

Techniques Used: Activity Assay, Indirect Immunoperoxidase Assay, Software, Activation Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Mouse Assay

TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P
Figure Legend Snippet: TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P

Techniques Used: Labeling, Enzyme-linked Immunosorbent Assay, Cell Culture, Mouse Assay, Plasmid Preparation

Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P
Figure Legend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P

Techniques Used: Mouse Assay, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P
Figure Legend Snippet: TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P

Techniques Used: Activation Assay, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Expressing, Mouse Assay, Plasmid Preparation, Injection

25) Product Images from "A benzenediamine derivate FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting myeloid dendritic cell-secreted BAFF"

Article Title: A benzenediamine derivate FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting myeloid dendritic cell-secreted BAFF

Journal: Acta Biochimica et Biophysica Sinica

doi: 10.1093/abbs/gmw017

FC-99 reduces the percentage, activation, and maturation of B cells in MRL/ lpr mice Serum levels of total IgG (A), IgG2a (B), and IgM (C) were determined by ELISA. (D) The percentage of B220 + B cells in spleens was analyzed by flow cytometry. (E and F)
Figure Legend Snippet: FC-99 reduces the percentage, activation, and maturation of B cells in MRL/ lpr mice Serum levels of total IgG (A), IgG2a (B), and IgM (C) were determined by ELISA. (D) The percentage of B220 + B cells in spleens was analyzed by flow cytometry. (E and F)

Techniques Used: Activation Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

26) Product Images from "Curcumin attenuates lupus nephritis in MRL/lpr mice by suppressing macrophage-secreted B cell activating factor (BAFF)"

Article Title: Curcumin attenuates lupus nephritis in MRL/lpr mice by suppressing macrophage-secreted B cell activating factor (BAFF)

Journal: International Journal of Clinical and Experimental Pathology

doi:

Curcumin attenuates lupus-like pathology in MRL/lpr mice. A. Proteinuria in MRL/lpr mice was determined using Mouse Albumin ELISA Quantitation Set; B. Serum levels of IgG against dsDNA in MRL/lpr mice were determined by ELISA; C. Serum levels of total IgG in MRL/ lpr mice were determined by ELISA; D. Kidney sections from MRL/ lpr lupus-prone mice showed histologic differences. H E staining revealed inflammatory infiltrates. IgG deposit was analyzed by IHC staining. The data represent the mean scores ± SEM ( *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 ).
Figure Legend Snippet: Curcumin attenuates lupus-like pathology in MRL/lpr mice. A. Proteinuria in MRL/lpr mice was determined using Mouse Albumin ELISA Quantitation Set; B. Serum levels of IgG against dsDNA in MRL/lpr mice were determined by ELISA; C. Serum levels of total IgG in MRL/ lpr mice were determined by ELISA; D. Kidney sections from MRL/ lpr lupus-prone mice showed histologic differences. H E staining revealed inflammatory infiltrates. IgG deposit was analyzed by IHC staining. The data represent the mean scores ± SEM ( *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 ).

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Staining, Immunohistochemistry

27) Product Images from "Curcumin attenuates lupus nephritis in MRL/lpr mice by suppressing macrophage-secreted B cell activating factor (BAFF)"

Article Title: Curcumin attenuates lupus nephritis in MRL/lpr mice by suppressing macrophage-secreted B cell activating factor (BAFF)

Journal: International Journal of Clinical and Experimental Pathology

doi:

Curcumin attenuates lupus-like pathology in MRL/lpr mice. A. Proteinuria in MRL/lpr mice was determined using Mouse Albumin ELISA Quantitation Set; B. Serum levels of IgG against dsDNA in MRL/lpr mice were determined by ELISA; C. Serum levels of total IgG in MRL/ lpr mice were determined by ELISA; D. Kidney sections from MRL/ lpr lupus-prone mice showed histologic differences. H E staining revealed inflammatory infiltrates. IgG deposit was analyzed by IHC staining. The data represent the mean scores ± SEM ( *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 ).
Figure Legend Snippet: Curcumin attenuates lupus-like pathology in MRL/lpr mice. A. Proteinuria in MRL/lpr mice was determined using Mouse Albumin ELISA Quantitation Set; B. Serum levels of IgG against dsDNA in MRL/lpr mice were determined by ELISA; C. Serum levels of total IgG in MRL/ lpr mice were determined by ELISA; D. Kidney sections from MRL/ lpr lupus-prone mice showed histologic differences. H E staining revealed inflammatory infiltrates. IgG deposit was analyzed by IHC staining. The data represent the mean scores ± SEM ( *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 ).

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Staining, Immunohistochemistry

28) Product Images from "Dichotomy in FcγRIIB deficiency and autoimmune-prone SLAM haplotype clarifies the roles of the Fc receptor in development of autoantibodies and glomerulonephritis"

Article Title: Dichotomy in FcγRIIB deficiency and autoimmune-prone SLAM haplotype clarifies the roles of the Fc receptor in development of autoantibodies and glomerulonephritis

Journal: BMC Immunology

doi: 10.1186/s12865-014-0047-y

Female-biased production of anti-nuclear antibodies in RIIB −/− mice. (A) The total IgM and IgG levels in sera from different lines, including B6, RIIB −/− , SLAM 129 , and RIIB −/− SLAM 129 , at 24–28 weeks of age of both genders were measured by ELISA. For IgM and IgG determinations in male B6 mice, n =3 and 5, respectively; for other determinations, n ≥8. The data in each panel are from two separate measurements and are presented as means ± S.D. * P
Figure Legend Snippet: Female-biased production of anti-nuclear antibodies in RIIB −/− mice. (A) The total IgM and IgG levels in sera from different lines, including B6, RIIB −/− , SLAM 129 , and RIIB −/− SLAM 129 , at 24–28 weeks of age of both genders were measured by ELISA. For IgM and IgG determinations in male B6 mice, n =3 and 5, respectively; for other determinations, n ≥8. The data in each panel are from two separate measurements and are presented as means ± S.D. * P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

29) Product Images from "Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals"

Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01364

Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P
Figure Legend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) inhibits mammalian target of rapamycin complex 1 (mTORC1) activity in lipopolysaccharide (LPS)-stimulated B cells. (A) The seven most related cellular functions and canonical pathways in genes of cluster 6 in Figure 4 A were analyzed by in Ingenuity Pathway Analysis (IPA) software. (B) Heatmap visualization of activation z -score ratios of LPS-stimulated B cells to LPS-stimulated B cells with IL-10/TGF-β3 less than −3.1 calculated by the upstream regulator analysis in IPA software. All of the 1,895 differentially expressed genes depicted in Figure 4 A were utilized in this analysis. (C) Representative western blot analyses of total and phosphorylated protein levels in LPS-stimulated B cells treated either with TGF-β3 and/or IL-10 for 24–48 h. (D) Flow cytometric (FCM) quantification of phosphorylated S6RP at Ser235/236 and 4E-BP1 at Thr37/46 in LPS-stimulated B cells either with or without TGF-β3 and/or IL-10 for 72 h ( n = 3). (E) Total IgG antibody titers in the supernatants of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 2 µM MHY1485 for 7 days quantified by ELISA ( n = 3). Data are representative of more than two independent experiments in (C–E) . (F) FCM quantification of phosphorylated S6RP at Ser235/236 in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). P

Techniques Used: Activity Assay, Indirect Immunoperoxidase Assay, Software, Activation Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Mouse Assay

TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P
Figure Legend Snippet: TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P

Techniques Used: Labeling, Enzyme-linked Immunosorbent Assay, Cell Culture, Mouse Assay, Plasmid Preparation

Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P
Figure Legend Snippet: Cytokine synergy of TGF-β3 and interleukin-10 (IL-10) regulates metabolism in toll-like receptor-stimulated B cells. (A) Splenic B cells from LC3-green fluorescent protein (GFP) mice were stimulated by lipopolysaccharides (LPS) either with TGF-β3 and/or IL-10 for 3 days. GFP − LC3 + spots captured by fluorescence microscopy were counted manually ( n = 20). (B) Relative Prdm1 expression in LPS-stimulated B cells either with 1 mM 2-DG, 10 mM sodium pyruvate, 0.5 µM rotenone (Rot)/antimycin A (AA), or 10 µM M1/Mdivi1 analyzed by qRT-PCR ( n = 3). (C) Flow cytometric analysis of MitoTracker-stained, LPS-stimulated B cells with each cytokine cultured for 3 days. (D,E) Oxygen consumption rate (OCR) (D) and ECAR (E) of LPS-stimulated B cells with each cytokine measured by extracellular flux analyzer ( n = 3). (F,G) IgG antibody titers quantified by ELISA (F) and OCR measured by extracellular analyzer (G) of LPS-stimulated B cells with or without TGF-β3 and IL-10 in the presence or absence of 10 µM M1/Mdivi1 ( n = 3). (H) Mean fluorescence intensity (MFI) of MitoTracker Green in splenic B220 + cells from B6 mice treated as in Figure 2 E ( n = 9–10). (I) Human B cells were stimulated by 2.5 µg/ml CpG-ODN, 1,000 U/ml IL-2, 10 ng/ml IL-6, and 0.5 µg/ml anti-CD40 with 10 pg/ml TGF-β3, 10 ng/ml IL-10, and/or 1 µg/ml anti-IL-10 Ab for 7 days ( n = 3). IgG2 antibody titers of the supernatants were quantified by ELISA. (J) OCR of CpG-ODN-stimulated human B cells with each cytokine for 3 days measured by extracellular flux analyzer ( n = 3). P

Techniques Used: Mouse Assay, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P
Figure Legend Snippet: TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P

Techniques Used: Activation Assay, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Expressing, Mouse Assay, Plasmid Preparation, Injection

30) Product Images from "Human Vγ9Vδ2-T Cells Synergize CD4+ T Follicular Helper Cells to Produce Influenza Virus-Specific Antibody"

Article Title: Human Vγ9Vδ2-T Cells Synergize CD4+ T Follicular Helper Cells to Produce Influenza Virus-Specific Antibody

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00599

Vγ9Vδ2-T cells increased H9N2 virus-specific IgG and IgM productions in vivo . Specific immune cells-depleted and whole peripheral blood mononuclear cells (PBMC)-humanized mice were vaccinated with UV-inactivated H9N2 virus through i.p. pathway on day 0 and 7. Serum was collected on day 21 post the first vaccination. (A,B) Total IgG and IgM in serum (C,D) H9N2 virus-specific IgG and IgM in serum that treated with receptor destroyed enzyme (RDE) were determined by enzyme-linked immunosorbent assay. (E) H9N2-specific antibodies in serum that treated with RDE were determined by hemagglutination inhibition assay. The data shown were the represent of four independent experiments. (F) Representative histological analysis, immunohistological stainings for human CD4 T, B, and γδ-T cells in formalin-fixed paraffin-embedded spleen in humanized mice constructed with whole PBMCs after UV-H9N2 virus immunization. Scale bars = 100 μm. Bottom panels showed the higher-magnification views of the respective boxed areas in the top panels. Scale bars = 50 μm. The data shown are the mean ± SEM. * p
Figure Legend Snippet: Vγ9Vδ2-T cells increased H9N2 virus-specific IgG and IgM productions in vivo . Specific immune cells-depleted and whole peripheral blood mononuclear cells (PBMC)-humanized mice were vaccinated with UV-inactivated H9N2 virus through i.p. pathway on day 0 and 7. Serum was collected on day 21 post the first vaccination. (A,B) Total IgG and IgM in serum (C,D) H9N2 virus-specific IgG and IgM in serum that treated with receptor destroyed enzyme (RDE) were determined by enzyme-linked immunosorbent assay. (E) H9N2-specific antibodies in serum that treated with RDE were determined by hemagglutination inhibition assay. The data shown were the represent of four independent experiments. (F) Representative histological analysis, immunohistological stainings for human CD4 T, B, and γδ-T cells in formalin-fixed paraffin-embedded spleen in humanized mice constructed with whole PBMCs after UV-H9N2 virus immunization. Scale bars = 100 μm. Bottom panels showed the higher-magnification views of the respective boxed areas in the top panels. Scale bars = 50 μm. The data shown are the mean ± SEM. * p

Techniques Used: In Vivo, Mouse Assay, Enzyme-linked Immunosorbent Assay, HI Assay, Formalin-fixed Paraffin-Embedded, Construct

Vγ9Vδ2-T cells facilitated influenza virus-specific antibody production. Naïve CD4 T cells were cultured with UV-H9N2 virus-pulsed dendritic cells for 4–5 days, then, the activated CD4 T cells were cultured with CD19 + B cells from same donor with or without Vγ9Vδ2-T cells for another 7–10 days. (A,B) Total IgG and IgM in supernatant on day 7 were detected by enzyme-linked immunosorbent assay (ELISA). (C,D) H9N2 virus-specific IgG and IgM were detected by ELISA on day 7. Each dot means one donor. The data shown are the mean ± SEM. * p
Figure Legend Snippet: Vγ9Vδ2-T cells facilitated influenza virus-specific antibody production. Naïve CD4 T cells were cultured with UV-H9N2 virus-pulsed dendritic cells for 4–5 days, then, the activated CD4 T cells were cultured with CD19 + B cells from same donor with or without Vγ9Vδ2-T cells for another 7–10 days. (A,B) Total IgG and IgM in supernatant on day 7 were detected by enzyme-linked immunosorbent assay (ELISA). (C,D) H9N2 virus-specific IgG and IgM were detected by ELISA on day 7. Each dot means one donor. The data shown are the mean ± SEM. * p

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

31) Product Images from "Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals"

Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01364

TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P
Figure Legend Snippet: TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P

Techniques Used: Labeling, Enzyme-linked Immunosorbent Assay, Cell Culture, Mouse Assay, Plasmid Preparation

TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P
Figure Legend Snippet: TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P

Techniques Used: Activation Assay, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Expressing, Mouse Assay, Plasmid Preparation, Injection

32) Product Images from "Collagen‐derived peptides modulate CD4+ T‐cell differentiation and suppress allergic responses in mice"

Article Title: Collagen‐derived peptides modulate CD4+ T‐cell differentiation and suppress allergic responses in mice

Journal: Immunity, Inflammation and Disease

doi: 10.1002/iid3.213

Effects of collagen‐peptide administration on humoral immune responses. BALB/c mice fed a diet with or without collagen peptides were intraperitoneally injected with 10 μg of OVA and Alum at weeks 1, 2, and 3. Serum was collected from each mouse, and total and OVA‐specific IgE and IgG were measured using ELISA. Values are means ± SEM ( n = 16) obtained by two independent experiments. ** P
Figure Legend Snippet: Effects of collagen‐peptide administration on humoral immune responses. BALB/c mice fed a diet with or without collagen peptides were intraperitoneally injected with 10 μg of OVA and Alum at weeks 1, 2, and 3. Serum was collected from each mouse, and total and OVA‐specific IgE and IgG were measured using ELISA. Values are means ± SEM ( n = 16) obtained by two independent experiments. ** P

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

33) Product Images from "Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals"

Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01364

TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P
Figure Legend Snippet: TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P

Techniques Used: Activation Assay, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Expressing, Mouse Assay, Plasmid Preparation, Injection

34) Product Images from "Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals"

Article Title: Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01364

TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P
Figure Legend Snippet: TGF-β and interleukin-10 (IL-10) synergistically suppress toll-like receptor-mediated humoral immune responses. (A) Histogram plots and percentages of CFSE-labeled B220 + B cells stimulated by 10 µg/ml lipopolysaccharides (LPS) either with or without 2 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 3 days ( n = 3). (B) Total IgG antibody titers in the supernatants of 3 µg/ml LPS-stimulated B cells either with 10 ng/ml TGF-β1, TGF-β3, and/or 50 ng/ml IL-10 for 7 days were quantified by ELISA ( n = 3). (C) B cells were cultured under 200 ng/ml R848 and anti-IgM stimulation either with or without TGF-β1 or TGF-β3 for 7 days and total IgG antibody production was assessed by ELISA ( n = 3). (D) Total IgG antibody titer in the supernatants of B cells cultured with LPS and 25 ng/ml IL-6 (left) or 1 ng/ml IL-17 (right) either with TGF-β1 or TGF-β3 for 7 days ( n = 3). Data are representative of more than two independent experiments in (A–D) . (E) Serum anti-NP-bovine serum albumin (BSA) antibody titers from NP-LPS-immunized B6 mice treated with 300 µg anti-IL-10 antibody and 300 µg anti-TGF-β antibody were quantified by ELISA ( n = 9–10). (F,G) Anti-NP-BSA antibody titers from NP-KLH/CFA-immunized ( n = 11–16) (F) or NP-LPS-immunized ( n = 9–10) (G) mice with indicated pCAGGS plasmid vectors were quantified. P

Techniques Used: Labeling, Enzyme-linked Immunosorbent Assay, Cell Culture, Mouse Assay, Plasmid Preparation

TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P
Figure Legend Snippet: TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml anti-CD40 and 10 µg/ml anti-IgM either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P

Techniques Used: Activation Assay, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Expressing, Mouse Assay, Plasmid Preparation, Injection

35) Product Images from "Collagen‐derived peptides modulate CD4+ T‐cell differentiation and suppress allergic responses in mice"

Article Title: Collagen‐derived peptides modulate CD4+ T‐cell differentiation and suppress allergic responses in mice

Journal: Immunity, Inflammation and Disease

doi: 10.1002/iid3.213

Effects of collagen‐peptide administration on humoral immune responses. BALB/c mice fed a diet with or without collagen peptides were intraperitoneally injected with 10 μg of OVA and Alum at weeks 1, 2, and 3. Serum was collected from each mouse, and total and OVA‐specific IgE and IgG were measured using ELISA. Values are means ± SEM ( n = 16) obtained by two independent experiments. ** P
Figure Legend Snippet: Effects of collagen‐peptide administration on humoral immune responses. BALB/c mice fed a diet with or without collagen peptides were intraperitoneally injected with 10 μg of OVA and Alum at weeks 1, 2, and 3. Serum was collected from each mouse, and total and OVA‐specific IgE and IgG were measured using ELISA. Values are means ± SEM ( n = 16) obtained by two independent experiments. ** P

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

36) Product Images from "Identification of Liver Epithelial Cell-derived Ig Expression in μ chain-deficient mice"

Article Title: Identification of Liver Epithelial Cell-derived Ig Expression in μ chain-deficient mice

Journal: Scientific Reports

doi: 10.1038/srep23669

IgM, IgG, IgD, IgA, Ig κ and Igλ detection in serum and liver cells. ( A ) Ig expression level in serum and liver lysate of WT mice and μMT mice were measured by Mouse Ig Isotyping Array. WT serum was diluted 100 times, while serum from μMT mice was diluted 50 times. The statistical analysis was performed under the diluted condition of serum and the dilution difference was not taken into account. Igs were detected in 30 μg liver protein from WT mice or μMT mice. ( B ) The concentrations of IgM in serum and liver lysate from WT mice and μMT mice were detected by ELISA. ***P
Figure Legend Snippet: IgM, IgG, IgD, IgA, Ig κ and Igλ detection in serum and liver cells. ( A ) Ig expression level in serum and liver lysate of WT mice and μMT mice were measured by Mouse Ig Isotyping Array. WT serum was diluted 100 times, while serum from μMT mice was diluted 50 times. The statistical analysis was performed under the diluted condition of serum and the dilution difference was not taken into account. Igs were detected in 30 μg liver protein from WT mice or μMT mice. ( B ) The concentrations of IgM in serum and liver lysate from WT mice and μMT mice were detected by ELISA. ***P

Techniques Used: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay

IgM and IgG were secreted from sorted liver epithelial cells. ( A ) CK18 was chosen as the sorting marker for liver epithelial cells. P1 represented the larger cells in liver. CK18 + cells in P1 gate, which were P2 cells, were harvested. ( B ) Secreted IgM and IgG of liver epithelial cells sorted by CK18 from WT mice and μMT mice were detected by ELISPOT. WT spleen cells were used as positive control.
Figure Legend Snippet: IgM and IgG were secreted from sorted liver epithelial cells. ( A ) CK18 was chosen as the sorting marker for liver epithelial cells. P1 represented the larger cells in liver. CK18 + cells in P1 gate, which were P2 cells, were harvested. ( B ) Secreted IgM and IgG of liver epithelial cells sorted by CK18 from WT mice and μMT mice were detected by ELISPOT. WT spleen cells were used as positive control.

Techniques Used: Marker, Mouse Assay, Enzyme-linked Immunospot, Positive Control

Identification of B cells and Igs in μMT mice. ( A ) B lymphocyte and T lymphocyte in peripheral blood of WT mice and μMT mice were detected with PE anti-mouse B220 and FITC anti-mouse CD3 by FACS. ( B ) Pro-B cell (CD43 + B220 low ) and pre-B cell (CD43 − B220 + ) in BM of WT mice and μMT mice were detected with PE anti-mouse CD43 and PE/Cy7 anti-mouse B220 by FACS. The cells in the upper left circle were pre-B cells, and the cells in the bottom right circle were pro-B cells. ( C ) Mature B cell (B220 + IgM + ) in BM of WT mice and μMT mice were detected with FITC anti-mouse IgM and PE/Cy7 anti-mouse B220 by FACS. The circle showed mature B cells. ( D ) The concentrations of IgM and IgG in serum from WT mice and μMT mice were detected by ELISA. ***P
Figure Legend Snippet: Identification of B cells and Igs in μMT mice. ( A ) B lymphocyte and T lymphocyte in peripheral blood of WT mice and μMT mice were detected with PE anti-mouse B220 and FITC anti-mouse CD3 by FACS. ( B ) Pro-B cell (CD43 + B220 low ) and pre-B cell (CD43 − B220 + ) in BM of WT mice and μMT mice were detected with PE anti-mouse CD43 and PE/Cy7 anti-mouse B220 by FACS. The cells in the upper left circle were pre-B cells, and the cells in the bottom right circle were pro-B cells. ( C ) Mature B cell (B220 + IgM + ) in BM of WT mice and μMT mice were detected with FITC anti-mouse IgM and PE/Cy7 anti-mouse B220 by FACS. The circle showed mature B cells. ( D ) The concentrations of IgM and IgG in serum from WT mice and μMT mice were detected by ELISA. ***P

Techniques Used: Mouse Assay, FACS, Enzyme-linked Immunosorbent Assay

37) Product Images from "CD180 Ligation Inhibits TLR7- and TLR9-Mediated Activation of Macrophages and Dendritic Cells Through the Lyn-SHP-1/2 Axis in Murine Lupus"

Article Title: CD180 Ligation Inhibits TLR7- and TLR9-Mediated Activation of Macrophages and Dendritic Cells Through the Lyn-SHP-1/2 Axis in Murine Lupus

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02643

Treatment effect of anti-CD180 Ab on lupus-symptoms of MRL/ lpr mice. (A) Proteinuria was collected weekly and determined by ELISA. (B) Representative images of marked splenomegaly in all groups of mice. (C,D) ELISA analysis of the anti-dsDNA antibody (C) and anti-RNA antibody (D) in serum. (E) H E staining of the kidney sections from all groups of mice. (F) Immunofluorescence staining to detect IgG deposit in the kidney sections. (G) Immunofluorescence staining to detect IgM deposit in the kidney sections. (H–J) Flow cytometric analysis of CD86 expression on peritoneal macrophages (H) , splenic macrophages (I) , and DCs (J) in all groups of mice. The data are shown as the means ± SEM ( n = 6 mice/group). ** p
Figure Legend Snippet: Treatment effect of anti-CD180 Ab on lupus-symptoms of MRL/ lpr mice. (A) Proteinuria was collected weekly and determined by ELISA. (B) Representative images of marked splenomegaly in all groups of mice. (C,D) ELISA analysis of the anti-dsDNA antibody (C) and anti-RNA antibody (D) in serum. (E) H E staining of the kidney sections from all groups of mice. (F) Immunofluorescence staining to detect IgG deposit in the kidney sections. (G) Immunofluorescence staining to detect IgM deposit in the kidney sections. (H–J) Flow cytometric analysis of CD86 expression on peritoneal macrophages (H) , splenic macrophages (I) , and DCs (J) in all groups of mice. The data are shown as the means ± SEM ( n = 6 mice/group). ** p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Flow Cytometry, Expressing

Treatment effect of anti-CD180 Ab on lupus-symptoms of IMQ-treated mice. (A) Treatment schematic of anti-CD180 Ab and IgG2b injection to mice following epicutaneous application of the TLR7 agonist imiquimod. (B) Proteinuria was collected weekly and determined by ELISA. (C) Representative images of marked splenomegaly in all groups of mice. (D,E) ELISA analysis of the anti-dsDNA antibody (D) and anti-RNA antibody (E) in serum. (F) H E staining of the kidney sections from all groups of mice. (G) Immunofluorescence staining to detect IgG deposit in the kidney sections. (H) Immunofluorescence staining to detect IgM deposit in the kidney sections. (I–K) Flow cytometric analysis of CD86 expression on peritoneal macrophages (I) , splenic macrophages (J) , and DCs (K) in all groups of mice. The data are shown as the means ± SEM ( n = 7 mice/group). * p
Figure Legend Snippet: Treatment effect of anti-CD180 Ab on lupus-symptoms of IMQ-treated mice. (A) Treatment schematic of anti-CD180 Ab and IgG2b injection to mice following epicutaneous application of the TLR7 agonist imiquimod. (B) Proteinuria was collected weekly and determined by ELISA. (C) Representative images of marked splenomegaly in all groups of mice. (D,E) ELISA analysis of the anti-dsDNA antibody (D) and anti-RNA antibody (E) in serum. (F) H E staining of the kidney sections from all groups of mice. (G) Immunofluorescence staining to detect IgG deposit in the kidney sections. (H) Immunofluorescence staining to detect IgM deposit in the kidney sections. (I–K) Flow cytometric analysis of CD86 expression on peritoneal macrophages (I) , splenic macrophages (J) , and DCs (K) in all groups of mice. The data are shown as the means ± SEM ( n = 7 mice/group). * p

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Flow Cytometry, Expressing

38) Product Images from "Collagen‐derived peptides modulate CD4+ T‐cell differentiation and suppress allergic responses in mice"

Article Title: Collagen‐derived peptides modulate CD4+ T‐cell differentiation and suppress allergic responses in mice

Journal: Immunity, Inflammation and Disease

doi: 10.1002/iid3.213

Effects of collagen‐peptide administration on humoral immune responses. BALB/c mice fed a diet with or without collagen peptides were intraperitoneally injected with 10 μg of OVA and Alum at weeks 1, 2, and 3. Serum was collected from each mouse, and total and OVA‐specific IgE and IgG were measured using ELISA. Values are means ± SEM ( n = 16) obtained by two independent experiments. ** P
Figure Legend Snippet: Effects of collagen‐peptide administration on humoral immune responses. BALB/c mice fed a diet with or without collagen peptides were intraperitoneally injected with 10 μg of OVA and Alum at weeks 1, 2, and 3. Serum was collected from each mouse, and total and OVA‐specific IgE and IgG were measured using ELISA. Values are means ± SEM ( n = 16) obtained by two independent experiments. ** P

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

39) Product Images from "Prevention and Treatment of Influenza with Hyperimmune Bovine Colostrum Antibody"

Article Title: Prevention and Treatment of Influenza with Hyperimmune Bovine Colostrum Antibody

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013622

Half-lives of intranasally-delivered bovine IgG in the nose, lungs and serum. The bovine anti-PR8 IgG preparation (1 mg) was administered in a volume of 50 µl by the i.n. route to anesthetized mice. At 1, 12, 24 and 36 hr post-administration, mice were killed and bovine IgG was detected by capture ELISA in nasal turbinate and lung homogenates and in sera. The total amount of Ab was determined by reference to a standard curve. Linear regression analysis was used to calculate the half-lives of bovine IgG in the different sites. The data are representative of two experiments.
Figure Legend Snippet: Half-lives of intranasally-delivered bovine IgG in the nose, lungs and serum. The bovine anti-PR8 IgG preparation (1 mg) was administered in a volume of 50 µl by the i.n. route to anesthetized mice. At 1, 12, 24 and 36 hr post-administration, mice were killed and bovine IgG was detected by capture ELISA in nasal turbinate and lung homogenates and in sera. The total amount of Ab was determined by reference to a standard curve. Linear regression analysis was used to calculate the half-lives of bovine IgG in the different sites. The data are representative of two experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

40) Product Images from "Collagen‐derived peptides modulate CD4+ T‐cell differentiation and suppress allergic responses in mice"

Article Title: Collagen‐derived peptides modulate CD4+ T‐cell differentiation and suppress allergic responses in mice

Journal: Immunity, Inflammation and Disease

doi: 10.1002/iid3.213

Effects of collagen‐peptide administration on humoral immune responses. BALB/c mice fed a diet with or without collagen peptides were intraperitoneally injected with 10 μg of OVA and Alum at weeks 1, 2, and 3. Serum was collected from each mouse, and total and OVA‐specific IgE and IgG were measured using ELISA. Values are means ± SEM ( n = 16) obtained by two independent experiments. ** P
Figure Legend Snippet: Effects of collagen‐peptide administration on humoral immune responses. BALB/c mice fed a diet with or without collagen peptides were intraperitoneally injected with 10 μg of OVA and Alum at weeks 1, 2, and 3. Serum was collected from each mouse, and total and OVA‐specific IgE and IgG were measured using ELISA. Values are means ± SEM ( n = 16) obtained by two independent experiments. ** P

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

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Enzyme-linked Immunosorbent Assay:

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Article Snippet: .. Enzyme-linked immunosorbent assay (ELISA) The concentrations of IgE, tumor necrosis factor (TNF)-α, and TSLP in the collected sera of the blood were determined by ELISA kit, according to the manufacturer's instructions (IgE; Bethyl Laboratories, Montgomery, TX, USA) and R & D Systems (TNF-α and TSLP). .. The optical density was measured at 450 nm using an ELISA reader (BMG Labtech, Baden-Württemberg, Germany).

Article Title: Thyrotrophin receptor-specific memory T cell responses require normal B cells in a murine model of Graves's disease
Article Snippet: .. IgM and IgG levels were measured using enzyme-linked immuno-sorbent assay (ELISA) quantification kits (Bethyl Laboratories Inc, Montgomery, TX, USA). .. The sera studied were obtained from mice euthanized 8 weeks after the third immunization with adenovirus (Ad–TSHR or Ad–Con).

Article Title: Plasmacytoid dendritic cell deficiency in neonates enhances allergic airway inflammation via reduced production of IFN-α
Article Snippet: .. IL-4, IL-5, IL-10, IL-17A, IFN-γ, GM-CSF, and IgE ELISA kits were from BioLegend (San Diego, CA); IL-13 ELISA kit was from eBioscience (San Diego, CA); IL-33 and CCL20 ELISA kits were from R & D System (Minneapolis, MN); and IgG1 and IgG2a ELISA kits were from Bethyl (Montgomery, TX). .. Mouse antibodies used for flow cytometry were anti-siglec-F (clone E50-2440; BD, San Jose, CA), anti-Ly6G (clone 1A8; BD), anti-CD11c (clone HL3; BD), anti-CD3e (clone 145-2C11; BD), anti-CD19 (clone 1D3; BD), anti-CD11b (clone M1/70; BD), anti-CD64 (clone X54-5/7.1; BD), anti-CD45 (clone 30-F11; BD), anti-CD90.2 (clone 53-2.1; BD), anti-CD25 (clone PC61; BD), anti-ST2 (clone U29-93; BD), anti-B220 (clone RA3-6B2; BD), anti-siglec-H (clone eBio440c; eBioscience), anti-IA/IE (clone 2G9; BD), anti-PDCA1 (clone eBio927; eBioscience), anti-CD103 (clone M290; BD), anti-CD4 (clone H129.19; BD), anti-FoxP3 (clone MF23; BD), anti-GARP (clone F011-5; BioLegend), anti-IL-10 (clone JES5-16E3; BD), anti-TGF-β (clone TW7-20B9; BioLegend), anti-CD326 (clone G8.8; BD), and anti-CD31 (clone MEC 13.3; BD).

other:

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Article Snippet: For protocol (b), the serum was diluted as follows: IgA 1 : 1000, IgG1 1 : 5000, IgG2a 1 : 5000, and IgE 1 : 100, and the BALF was diluted as follows: IgA 1 : 10, IgG1 1 : 20, IgG2a 1 : 20, and IgE 1 : 2.

Cell Culture:

Article Title: Structural and molecular basis for hyperspecificity of RNA aptamer to human immunoglobulin G
Article Snippet: .. Human IgG1 through IgG4 were purchased from Calbiochem; mouse IgG1 and IgG2a, human serum, and mouse serum from Chemicon; mouse IgG2b, rat IgG2a, rat IgG2b, and rabbit IgG from Zymed Laboratories; mouse IgG3, bovine IgG1, bovine IgG2, and cat IgG from Bethyl; rat IgG1, RANK-Fc fusion protein (hIgG1-Fc Pro100-Lys330) and CD28-Fc fusion protein from R & D Systems; IgGs from chicken and dog from Rockland; rat IgG2c from UK-Seratec; guinea pig IgG from Biogenesis; Remicade (Infliximab, 10 mg/mL) from Centocor B.V. Anti-CD26 humanized antibody was a kind gift from Chikao Morimoto (Institute of Medical Science, University of Tokyo), and CHO cell culture fluid containing CamPath (Alemtuzumab, anti-CD52 humanized antibody) was a kind gift from G. Hale and H. Waldmann (the Therapeutic Antibody Center, Oxford). rProtein A-conjugated resin and NHS-activated Sepharose 4 resin were purchased from Amersham Bioscience, Tresyl-TOYOPEARL resin from TOSOH, Ni-NTA affinity resin from Qiagen, and BD TalonRT affinity resin from BD science. .. RNA was prepared either by in vitro transcription using a DuraScribe T7 Transcription kit (Epicentre) according to the manufacturer's instructions or by chemical synthesis (Gene Design, Inc.).

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    Bethyl mouse igg elisa quantitation set
    In vitro immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on the Ig secretion by naive B cells induced by CpG ODNs. Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) CpG ODNs (1 µg/mL). After seven days of incubation, supernatants were collected and total IgM ( A ) and <t>IgG</t> ( B ) were quantified by <t>ELISA.</t> Cells stimulated with CpG ODNs alone (black bars) served as the positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of stimulation by CpG ODNs. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panel) experiments.
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    In vitro immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on the Ig secretion by naive B cells induced by CpG ODNs. Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) CpG ODNs (1 µg/mL). After seven days of incubation, supernatants were collected and total IgM ( A ) and IgG ( B ) were quantified by ELISA. Cells stimulated with CpG ODNs alone (black bars) served as the positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of stimulation by CpG ODNs. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panel) experiments.

    Journal: Pathogens

    Article Title: Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response

    doi: 10.3390/pathogens6020016

    Figure Lengend Snippet: In vitro immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on the Ig secretion by naive B cells induced by CpG ODNs. Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) CpG ODNs (1 µg/mL). After seven days of incubation, supernatants were collected and total IgM ( A ) and IgG ( B ) were quantified by ELISA. Cells stimulated with CpG ODNs alone (black bars) served as the positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of stimulation by CpG ODNs. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panel) experiments.

    Article Snippet: Samples from In Vitro Assays The total IgM and IgG levels in B cell culture supernatants were measured with a Mouse IgM or a Mouse IgG ELISA Quantitation Set (Bethyl Laboratories, Montgomery, TX, USA), respectively, according to the manufacturer’s instructions.

    Techniques: In Vitro, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Positive Control

    Titration of capsular polysaccharide (CPS)-specific antibodies in mice after infection with S. suis serotype 2, S. suis serotype 14, group B Streptococcus (GBS) serotype III or GBS serotype V. Mice were infected with 2 × 10 7 CFU of live S. suis serotype 2 strain P1/7 ( n = 60) ( A ); 5 × 10 6 CFU of live S. suis serotype 14 strain DAN13730 ( n = 40) ( B ); 2 × 10 6 CFU of live GBS serotype III strain COH-1 ( n = 40) ( C ); or 10 4 CFU of live GBS serotype V strain CJB111 ( n = 40) ( D ). Total Ig (IgG plus IgM) anti-CPS titers were determined by ELISA on Days 7 (in blue), 14 (in green) and 21 (in red) in surviving mice. IgM and IgG anti-CPS titers were determined on Day 21 (in red). For comparative purpose, total Ig (IgG plus IgM), IgM and IgG anti-protein (‘prot’) titers were also determined on Day 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. “C − ” represents a pool of control mice ( n = 3) injected with vehicle solution, whose titers were evaluated on Days 7, 14 and 21. * Statistically significant difference ( p

    Journal: Pathogens

    Article Title: Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response

    doi: 10.3390/pathogens6020016

    Figure Lengend Snippet: Titration of capsular polysaccharide (CPS)-specific antibodies in mice after infection with S. suis serotype 2, S. suis serotype 14, group B Streptococcus (GBS) serotype III or GBS serotype V. Mice were infected with 2 × 10 7 CFU of live S. suis serotype 2 strain P1/7 ( n = 60) ( A ); 5 × 10 6 CFU of live S. suis serotype 14 strain DAN13730 ( n = 40) ( B ); 2 × 10 6 CFU of live GBS serotype III strain COH-1 ( n = 40) ( C ); or 10 4 CFU of live GBS serotype V strain CJB111 ( n = 40) ( D ). Total Ig (IgG plus IgM) anti-CPS titers were determined by ELISA on Days 7 (in blue), 14 (in green) and 21 (in red) in surviving mice. IgM and IgG anti-CPS titers were determined on Day 21 (in red). For comparative purpose, total Ig (IgG plus IgM), IgM and IgG anti-protein (‘prot’) titers were also determined on Day 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. “C − ” represents a pool of control mice ( n = 3) injected with vehicle solution, whose titers were evaluated on Days 7, 14 and 21. * Statistically significant difference ( p

    Article Snippet: Samples from In Vitro Assays The total IgM and IgG levels in B cell culture supernatants were measured with a Mouse IgM or a Mouse IgG ELISA Quantitation Set (Bethyl Laboratories, Montgomery, TX, USA), respectively, according to the manufacturer’s instructions.

    Techniques: Titration, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Injection

    Titration of CPS-specific antibodies in mice after immunization with purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS. Mice ( n = 8) were immunized with 2 µg of purified native or desialylated S. suis serotype 2 ( A ); S. suis serotype 14 ( B ); GBS serotype III ( C ); or GBS serotype V ( D ) CPS emulsified with STIMUNE ® . Total Ig (IgG plus IgM) anti-native CPS titers were determined by ELISA on Days 7, 14 and 21. “C − ” represents a pool of control mice ( n = 3) injected with STIMUNE ® only, whose titers were evaluated on Days 7, 14 and 21. Data from individual mice are presented, including the geometric mean with 95% confidence interval. * Statistically significant difference ( p

    Journal: Pathogens

    Article Title: Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response

    doi: 10.3390/pathogens6020016

    Figure Lengend Snippet: Titration of CPS-specific antibodies in mice after immunization with purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS. Mice ( n = 8) were immunized with 2 µg of purified native or desialylated S. suis serotype 2 ( A ); S. suis serotype 14 ( B ); GBS serotype III ( C ); or GBS serotype V ( D ) CPS emulsified with STIMUNE ® . Total Ig (IgG plus IgM) anti-native CPS titers were determined by ELISA on Days 7, 14 and 21. “C − ” represents a pool of control mice ( n = 3) injected with STIMUNE ® only, whose titers were evaluated on Days 7, 14 and 21. Data from individual mice are presented, including the geometric mean with 95% confidence interval. * Statistically significant difference ( p

    Article Snippet: Samples from In Vitro Assays The total IgM and IgG levels in B cell culture supernatants were measured with a Mouse IgM or a Mouse IgG ELISA Quantitation Set (Bethyl Laboratories, Montgomery, TX, USA), respectively, according to the manufacturer’s instructions.

    Techniques: Titration, Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay, Injection

    Adjuvant effect of CpG oligodeoxynucleotides (ODNs) on the CPS-specific humoral response in mice immunized with purified native or desialylated S. suis serotype 2 CPS or purified S. pneumoniae serotype 3 CPS (PS3). ( A ) In a first set of experiments, mice ( n = 8) were immunized with 2 µg of purified native (n) or desialylated (dS) S. suis serotype 2 CPS (CPS S. suis ) or PS3 in PBS on Day 0 and 80 µg of CpG ODNs two days after. The control group ( n = 8) received CPS or PS3 on Day 0 and PBS two days later. The placebo group ( n = 3) received PBS or CpG ODNs only. Total Ig (IgG plus IgM) anti-native S. suis type 2 CPS or anti-PS3 titers were determined by ELISA on Days 7, 14 and 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. ( B ) In a second set of experiments, mice ( n = 5) were immunized as described in (A), but splenocytes were collected on Day 5 after immunization, and anti-CPS antibody-secreting cells (ASCs) were enumerated by ELISpot as described in the Materials and Methods section. Data are expressed as arithmetic means with SEM. ( C ) Visualization of PS3 (top) or native S. suis type 2 CPS (bottom) specific ASCs in ELISpot wells from splenocytes of mice obtained in (B). * p

    Journal: Pathogens

    Article Title: Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response

    doi: 10.3390/pathogens6020016

    Figure Lengend Snippet: Adjuvant effect of CpG oligodeoxynucleotides (ODNs) on the CPS-specific humoral response in mice immunized with purified native or desialylated S. suis serotype 2 CPS or purified S. pneumoniae serotype 3 CPS (PS3). ( A ) In a first set of experiments, mice ( n = 8) were immunized with 2 µg of purified native (n) or desialylated (dS) S. suis serotype 2 CPS (CPS S. suis ) or PS3 in PBS on Day 0 and 80 µg of CpG ODNs two days after. The control group ( n = 8) received CPS or PS3 on Day 0 and PBS two days later. The placebo group ( n = 3) received PBS or CpG ODNs only. Total Ig (IgG plus IgM) anti-native S. suis type 2 CPS or anti-PS3 titers were determined by ELISA on Days 7, 14 and 21. Data are presented in a box-and-whiskers diagram with the ends of whiskers representing the minimum and the maximum value. ( B ) In a second set of experiments, mice ( n = 5) were immunized as described in (A), but splenocytes were collected on Day 5 after immunization, and anti-CPS antibody-secreting cells (ASCs) were enumerated by ELISpot as described in the Materials and Methods section. Data are expressed as arithmetic means with SEM. ( C ) Visualization of PS3 (top) or native S. suis type 2 CPS (bottom) specific ASCs in ELISpot wells from splenocytes of mice obtained in (B). * p

    Article Snippet: Samples from In Vitro Assays The total IgM and IgG levels in B cell culture supernatants were measured with a Mouse IgM or a Mouse IgG ELISA Quantitation Set (Bethyl Laboratories, Montgomery, TX, USA), respectively, according to the manufacturer’s instructions.

    Techniques: Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

    In vivo immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on ovalbumin (OVA)-specific antibody response. Mice ( n = 8) were co-immunized intraperitoneally with 10 µg of OVA and 2 µg of purified native S. suis serotype 2 ( A ), S. suis serotype 14 ( B ), GBS serotype III ( C ) or GBS serotype V ( D ) CPS in PBS on Days 0 and 21. Control group ( n = 8) received OVA only on Days 0 and 21. Total Ig (IgG plus IgM) anti-OVA titers were determined by ELISA on Days 7, 14, 21 and 35. “C − ” represents a pool of placebo mice ( n = 3) injected with PBS, whose titers were evaluated on Days 7, 14, 21 and 35. Data from individual mice are presented, including the geometric mean with 95% confidence interval. An arrow indicates secondary immunization. * p

    Journal: Pathogens

    Article Title: Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response

    doi: 10.3390/pathogens6020016

    Figure Lengend Snippet: In vivo immunomodulatory effect of purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on ovalbumin (OVA)-specific antibody response. Mice ( n = 8) were co-immunized intraperitoneally with 10 µg of OVA and 2 µg of purified native S. suis serotype 2 ( A ), S. suis serotype 14 ( B ), GBS serotype III ( C ) or GBS serotype V ( D ) CPS in PBS on Days 0 and 21. Control group ( n = 8) received OVA only on Days 0 and 21. Total Ig (IgG plus IgM) anti-OVA titers were determined by ELISA on Days 7, 14, 21 and 35. “C − ” represents a pool of placebo mice ( n = 3) injected with PBS, whose titers were evaluated on Days 7, 14, 21 and 35. Data from individual mice are presented, including the geometric mean with 95% confidence interval. An arrow indicates secondary immunization. * p

    Article Snippet: Samples from In Vitro Assays The total IgM and IgG levels in B cell culture supernatants were measured with a Mouse IgM or a Mouse IgG ELISA Quantitation Set (Bethyl Laboratories, Montgomery, TX, USA), respectively, according to the manufacturer’s instructions.

    Techniques: In Vivo, Purification, Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection

    In vitro immunomodulatory effect of purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on BAFF/IL-4-induced Ig secretion by naive B cells. ( A , B ) Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) BAFF (1 µg/mL) along with IL-4 (50 ng/mL); ( C , D ) in order to evaluate the influence of sialic acid, cells were also incubated with desialylated (dS) S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS in parallel to cells incubated with the respective native CPS (n) as described in ( A , B ). After seven days of incubation, supernatants were collected, and total IgM ( A , C ) and IgG ( B , D ) was quantified by ELISA. Cells stimulated with BAFF/IL-4 alone (black bars) served as a positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of BAFF/IL-4 stimulation. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panels) experiments. * p

    Journal: Pathogens

    Article Title: Evaluation of the Immunomodulatory Properties of Streptococcus suis and Group B Streptococcus Capsular Polysaccharides on the Humoral Response

    doi: 10.3390/pathogens6020016

    Figure Lengend Snippet: In vitro immunomodulatory effect of purified native or desialylated S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS on BAFF/IL-4-induced Ig secretion by naive B cells. ( A , B ) Mouse splenic B cells (10 6 cells/mL) were incubated with purified native S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS (each at 20 µg/mL) simultaneously with (central panel) or 24 h before the addition of (right panel) BAFF (1 µg/mL) along with IL-4 (50 ng/mL); ( C , D ) in order to evaluate the influence of sialic acid, cells were also incubated with desialylated (dS) S. suis serotype 2, S. suis serotype 14, GBS serotype III or GBS serotype V CPS in parallel to cells incubated with the respective native CPS (n) as described in ( A , B ). After seven days of incubation, supernatants were collected, and total IgM ( A , C ) and IgG ( B , D ) was quantified by ELISA. Cells stimulated with BAFF/IL-4 alone (black bars) served as a positive control. In the left panel, cells stimulated with medium (white bars) or each purified CPS alone are represented as an indication of the basal Ig secretion level of cells in absence of BAFF/IL-4 stimulation. Data are expressed as arithmetic means with the SEM of three (central and right panels) or four (left panels) experiments. * p

    Article Snippet: Samples from In Vitro Assays The total IgM and IgG levels in B cell culture supernatants were measured with a Mouse IgM or a Mouse IgG ELISA Quantitation Set (Bethyl Laboratories, Montgomery, TX, USA), respectively, according to the manufacturer’s instructions.

    Techniques: In Vitro, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Positive Control

    Female-biased production of anti-nuclear antibodies in RIIB −/− mice. (A) The total IgM and IgG levels in sera from different lines, including B6, RIIB −/− , SLAM 129 , and RIIB −/− SLAM 129 , at 24–28 weeks of age of both genders were measured by ELISA. For IgM and IgG determinations in male B6 mice, n =3 and 5, respectively; for other determinations, n ≥8. The data in each panel are from two separate measurements and are presented as means ± S.D. * P

    Journal: BMC Immunology

    Article Title: Dichotomy in FcγRIIB deficiency and autoimmune-prone SLAM haplotype clarifies the roles of the Fc receptor in development of autoantibodies and glomerulonephritis

    doi: 10.1186/s12865-014-0047-y

    Figure Lengend Snippet: Female-biased production of anti-nuclear antibodies in RIIB −/− mice. (A) The total IgM and IgG levels in sera from different lines, including B6, RIIB −/− , SLAM 129 , and RIIB −/− SLAM 129 , at 24–28 weeks of age of both genders were measured by ELISA. For IgM and IgG determinations in male B6 mice, n =3 and 5, respectively; for other determinations, n ≥8. The data in each panel are from two separate measurements and are presented as means ± S.D. * P

    Article Snippet: The total IgM and IgG levels were measured with a Mouse IgM ELISA Quantitation Set and a Mouse IgG ELISA Quantitation Set (Bethyl Laboratory, Inc.), respectively, according to the manufacturer’s protocols.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    CD74 deficiency improves cGVH-induced SLE-like pathology in mice. A. Kidney PAS staining and glomerulus and tubulointerstitial scores. B. Kidney IgG and C3 immunoflorescent staining and glomerulus scores. C. Kidney Mac-2, MCP-1, and MHC-II immunostaining. D. ELISA determined serum IL-2, IL-6, IL-17A, and TGF-β levels in both Cd74 +/+ and Cd74 −/− mice (both on B6 background) at 10 weeks after intraperitoneal transfer of splenocytes from B6 or bm12 mice. The number of mice per group is indicated in each bar. Representative figures in panels A to C are shown to the left. Scale: 50 μm.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: CD74 deficiency mitigates systemic lupus erythematosus-like autoimmunity and pathological findings in mice

    doi: 10.4049/jimmunol.1600028

    Figure Lengend Snippet: CD74 deficiency improves cGVH-induced SLE-like pathology in mice. A. Kidney PAS staining and glomerulus and tubulointerstitial scores. B. Kidney IgG and C3 immunoflorescent staining and glomerulus scores. C. Kidney Mac-2, MCP-1, and MHC-II immunostaining. D. ELISA determined serum IL-2, IL-6, IL-17A, and TGF-β levels in both Cd74 +/+ and Cd74 −/− mice (both on B6 background) at 10 weeks after intraperitoneal transfer of splenocytes from B6 or bm12 mice. The number of mice per group is indicated in each bar. Representative figures in panels A to C are shown to the left. Scale: 50 μm.

    Article Snippet: The ELISA kits used in this study include: mouse IL6 DuoSet (DY406, R & D Systems, Minneapolis, MN), mouse IL2 DuoSet (DY402, R & D Systems), mouse TGF-β ELISA ready-SET-Go (88-8350, eBioscience, San Diego, CA), mouse IL17A Platinum ELISA (BMS6001, eBioscience), mouse IgG ELISA Quantitation Set (E90-131, Bethyl Laboratories, Inc., Montgomery, TX), and mouse IgG1 ELISA Quantitation Set (E90-105, Bethyl Laboratories, Inc).

    Techniques: Mouse Assay, Staining, Immunostaining, Enzyme-linked Immunosorbent Assay

    CD74 deficiency mitigates SLE-like pathology in SLE-prone Fas lpr mice. A. Kidney PAS staining and glomerulus and tubulointerstitial scores; B. Kidney IgG and C3 immunofluorescent staining and glomerulus scores. C. Kidney Mac-2, MCP-1, and MHC-II immunostaining. D. ELISA determined serum IL6, IL17A, IL2, and TGF-β levels from 24-week-old Fas lpr Cd74 +/+ and Fas lpr Cd74 −/− mice. The number of mice per group is indicated in each parenthesis. Representative graphs for A – C are shown to the left of each panel. Scale: 50 μm.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: CD74 deficiency mitigates systemic lupus erythematosus-like autoimmunity and pathological findings in mice

    doi: 10.4049/jimmunol.1600028

    Figure Lengend Snippet: CD74 deficiency mitigates SLE-like pathology in SLE-prone Fas lpr mice. A. Kidney PAS staining and glomerulus and tubulointerstitial scores; B. Kidney IgG and C3 immunofluorescent staining and glomerulus scores. C. Kidney Mac-2, MCP-1, and MHC-II immunostaining. D. ELISA determined serum IL6, IL17A, IL2, and TGF-β levels from 24-week-old Fas lpr Cd74 +/+ and Fas lpr Cd74 −/− mice. The number of mice per group is indicated in each parenthesis. Representative graphs for A – C are shown to the left of each panel. Scale: 50 μm.

    Article Snippet: The ELISA kits used in this study include: mouse IL6 DuoSet (DY406, R & D Systems, Minneapolis, MN), mouse IL2 DuoSet (DY402, R & D Systems), mouse TGF-β ELISA ready-SET-Go (88-8350, eBioscience, San Diego, CA), mouse IL17A Platinum ELISA (BMS6001, eBioscience), mouse IgG ELISA Quantitation Set (E90-131, Bethyl Laboratories, Inc., Montgomery, TX), and mouse IgG1 ELISA Quantitation Set (E90-105, Bethyl Laboratories, Inc).

    Techniques: Mouse Assay, Staining, Immunostaining, Enzyme-linked Immunosorbent Assay

    IgM, IgG, IgD, IgA, Ig κ and Igλ detection in serum and liver cells. ( A ) Ig expression level in serum and liver lysate of WT mice and μMT mice were measured by Mouse Ig Isotyping Array. WT serum was diluted 100 times, while serum from μMT mice was diluted 50 times. The statistical analysis was performed under the diluted condition of serum and the dilution difference was not taken into account. Igs were detected in 30 μg liver protein from WT mice or μMT mice. ( B ) The concentrations of IgM in serum and liver lysate from WT mice and μMT mice were detected by ELISA. ***P

    Journal: Scientific Reports

    Article Title: Identification of Liver Epithelial Cell-derived Ig Expression in μ chain-deficient mice

    doi: 10.1038/srep23669

    Figure Lengend Snippet: IgM, IgG, IgD, IgA, Ig κ and Igλ detection in serum and liver cells. ( A ) Ig expression level in serum and liver lysate of WT mice and μMT mice were measured by Mouse Ig Isotyping Array. WT serum was diluted 100 times, while serum from μMT mice was diluted 50 times. The statistical analysis was performed under the diluted condition of serum and the dilution difference was not taken into account. Igs were detected in 30 μg liver protein from WT mice or μMT mice. ( B ) The concentrations of IgM in serum and liver lysate from WT mice and μMT mice were detected by ELISA. ***P

    Article Snippet: ELISA To detect the concentration of IgM and IgG in liver lysate of mice liver and serum, we used Mouse IgM ELISA Quantitation Set and Mouse IgG ELISA Quantitation Set (Bethyl Laboratories, USA).

    Techniques: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Identification of B cells and Igs in μMT mice. ( A ) B lymphocyte and T lymphocyte in peripheral blood of WT mice and μMT mice were detected with PE anti-mouse B220 and FITC anti-mouse CD3 by FACS. ( B ) Pro-B cell (CD43 + B220 low ) and pre-B cell (CD43 − B220 + ) in BM of WT mice and μMT mice were detected with PE anti-mouse CD43 and PE/Cy7 anti-mouse B220 by FACS. The cells in the upper left circle were pre-B cells, and the cells in the bottom right circle were pro-B cells. ( C ) Mature B cell (B220 + IgM + ) in BM of WT mice and μMT mice were detected with FITC anti-mouse IgM and PE/Cy7 anti-mouse B220 by FACS. The circle showed mature B cells. ( D ) The concentrations of IgM and IgG in serum from WT mice and μMT mice were detected by ELISA. ***P

    Journal: Scientific Reports

    Article Title: Identification of Liver Epithelial Cell-derived Ig Expression in μ chain-deficient mice

    doi: 10.1038/srep23669

    Figure Lengend Snippet: Identification of B cells and Igs in μMT mice. ( A ) B lymphocyte and T lymphocyte in peripheral blood of WT mice and μMT mice were detected with PE anti-mouse B220 and FITC anti-mouse CD3 by FACS. ( B ) Pro-B cell (CD43 + B220 low ) and pre-B cell (CD43 − B220 + ) in BM of WT mice and μMT mice were detected with PE anti-mouse CD43 and PE/Cy7 anti-mouse B220 by FACS. The cells in the upper left circle were pre-B cells, and the cells in the bottom right circle were pro-B cells. ( C ) Mature B cell (B220 + IgM + ) in BM of WT mice and μMT mice were detected with FITC anti-mouse IgM and PE/Cy7 anti-mouse B220 by FACS. The circle showed mature B cells. ( D ) The concentrations of IgM and IgG in serum from WT mice and μMT mice were detected by ELISA. ***P

    Article Snippet: ELISA To detect the concentration of IgM and IgG in liver lysate of mice liver and serum, we used Mouse IgM ELISA Quantitation Set and Mouse IgG ELISA Quantitation Set (Bethyl Laboratories, USA).

    Techniques: Mouse Assay, FACS, Enzyme-linked Immunosorbent Assay