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Role of GSTP in LPS- or IKKβ- mediated activation of NF-κB and production of pro-inflammatory cytokines: (A) <t>C10</t> lung epithelial cells were either transfected with WT HA-IKKβ or empty vector (EV) alone or in the presence of either FLAG-tagged WT GSTP or FLAG tagged Y7F mutant GSTP. Overexpression of IKKβ and GSTP were assessed via Western Blotting. (B) Assessment of GSTP enzymatic activity in cells overexpressing either WT FLAG-GSTP or Y7F FLAG-GSTP, or empty vector (-). GSTP enzymatic activity was assessed by detection of GSH conjugated CDNB. Assessment of basal or LPS-induced NF-κB luciferase activity (C) and <t>GM-CSF</t> levels in cell supernatants (D) following transfection with WT GSTP or Y7F GSTP. * p
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1) Product Images from "Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells"

Article Title: Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells

Journal: Redox Biology

doi: 10.1016/j.redox.2016.03.005

Role of GSTP in LPS- or IKKβ- mediated activation of NF-κB and production of pro-inflammatory cytokines: (A) C10 lung epithelial cells were either transfected with WT HA-IKKβ or empty vector (EV) alone or in the presence of either FLAG-tagged WT GSTP or FLAG tagged Y7F mutant GSTP. Overexpression of IKKβ and GSTP were assessed via Western Blotting. (B) Assessment of GSTP enzymatic activity in cells overexpressing either WT FLAG-GSTP or Y7F FLAG-GSTP, or empty vector (-). GSTP enzymatic activity was assessed by detection of GSH conjugated CDNB. Assessment of basal or LPS-induced NF-κB luciferase activity (C) and GM-CSF levels in cell supernatants (D) following transfection with WT GSTP or Y7F GSTP. * p
Figure Legend Snippet: Role of GSTP in LPS- or IKKβ- mediated activation of NF-κB and production of pro-inflammatory cytokines: (A) C10 lung epithelial cells were either transfected with WT HA-IKKβ or empty vector (EV) alone or in the presence of either FLAG-tagged WT GSTP or FLAG tagged Y7F mutant GSTP. Overexpression of IKKβ and GSTP were assessed via Western Blotting. (B) Assessment of GSTP enzymatic activity in cells overexpressing either WT FLAG-GSTP or Y7F FLAG-GSTP, or empty vector (-). GSTP enzymatic activity was assessed by detection of GSH conjugated CDNB. Assessment of basal or LPS-induced NF-κB luciferase activity (C) and GM-CSF levels in cell supernatants (D) following transfection with WT GSTP or Y7F GSTP. * p

Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Mutagenesis, Over Expression, Western Blot, Activity Assay, Luciferase

2) Product Images from "Netrin G1 promotes pancreatic tumorigenesis through cancer associated fibroblast driven nutritional support and immunosuppression"

Article Title: Netrin G1 promotes pancreatic tumorigenesis through cancer associated fibroblast driven nutritional support and immunosuppression

Journal: bioRxiv

doi: 10.1101/330209

NetG1 + CAFs create an immunosuppressive microenvironment that protects PDAC cells from NK cell induced death. A. Quantification of U-Plex (multiplex ELISA; GM-CSF, IL-1β, CCL20, IL-6, IL-8) and ELISAs (IL-15, TGF-β) of assorted cytokines with immunomodulatory or immunoattractive potentials, detected in the CM of TA, CON CAFs, or NetG1 KO CAFs, growing in 3D. N= 6 biological replicates. * compared to TA. B. Quantification of the % of NK-92 cells positive for markers of activation (IFNγ and Granzyme B) determined by flow cytometry after IL-2 pre-activated NK-92 cells (8×10 4 ) were in direct co-culture (CC) with CON or NetG1 KO CAFs (2×10 4 ) or treated with their conditioned media (CM) for 16 hours. * compared to CON CAF CC. C. Primary NK cells (10 5 ) were isolated from healthy human donors, pre-activated with IL-2/IL-12, incubated with CM from CON or NetG1 KO CAFs for 16 hours, and their activation status was determined by flow cytometry, using IFNγ and CD69 as markers. Expression of markers was normalized to the positive control (IL-2 alone = 1.0). * comparison between the CON and KO at each % of CM. D-E. RFP + CON or NGL-1 KO PDACc (2×10 4 ) were co-cultured in 3D with GFP + CON or NetG1 KO CAFs (2×10 4 ) and with active (D) or resting (E) NK-92 cells (8×10 4 ) for 48 hours and PDAC survival was quantified. Groups were normalized to CON PDACc/CON CAF with active NK cells. Dotted line in the resting graph (RIGHT) denotes PDACc survival with CON PDACc/CON CAF with active NK cells. * compared to CON PDACc/CON CAF with active NK cells. One-Way ANOVA, Dunnett’s multiple comparison test (A, B, D) or T-test (C). *p
Figure Legend Snippet: NetG1 + CAFs create an immunosuppressive microenvironment that protects PDAC cells from NK cell induced death. A. Quantification of U-Plex (multiplex ELISA; GM-CSF, IL-1β, CCL20, IL-6, IL-8) and ELISAs (IL-15, TGF-β) of assorted cytokines with immunomodulatory or immunoattractive potentials, detected in the CM of TA, CON CAFs, or NetG1 KO CAFs, growing in 3D. N= 6 biological replicates. * compared to TA. B. Quantification of the % of NK-92 cells positive for markers of activation (IFNγ and Granzyme B) determined by flow cytometry after IL-2 pre-activated NK-92 cells (8×10 4 ) were in direct co-culture (CC) with CON or NetG1 KO CAFs (2×10 4 ) or treated with their conditioned media (CM) for 16 hours. * compared to CON CAF CC. C. Primary NK cells (10 5 ) were isolated from healthy human donors, pre-activated with IL-2/IL-12, incubated with CM from CON or NetG1 KO CAFs for 16 hours, and their activation status was determined by flow cytometry, using IFNγ and CD69 as markers. Expression of markers was normalized to the positive control (IL-2 alone = 1.0). * comparison between the CON and KO at each % of CM. D-E. RFP + CON or NGL-1 KO PDACc (2×10 4 ) were co-cultured in 3D with GFP + CON or NetG1 KO CAFs (2×10 4 ) and with active (D) or resting (E) NK-92 cells (8×10 4 ) for 48 hours and PDAC survival was quantified. Groups were normalized to CON PDACc/CON CAF with active NK cells. Dotted line in the resting graph (RIGHT) denotes PDACc survival with CON PDACc/CON CAF with active NK cells. * compared to CON PDACc/CON CAF with active NK cells. One-Way ANOVA, Dunnett’s multiple comparison test (A, B, D) or T-test (C). *p

Techniques Used: Multiplex Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Flow Cytometry, Co-Culture Assay, Isolation, Incubation, Expressing, Positive Control, Cell Culture

Knockdown of NetG1 by CRISPRi in two patient CAF lines results in a loss of pro-tumor immunosuppressive capacities. A. CON CAFs or NetG1 KD CAFs were allowed to produce CM for 48 hours in 3D. The CM was then subjected to ELISA analysis for the quantification of the following cytokines: GM-CSF, IL-6, IL-8, TGF-β, and IL-15. N= 3 biological replicates. One-way ANOVA, Dunnett’s multiple comparison test, * p
Figure Legend Snippet: Knockdown of NetG1 by CRISPRi in two patient CAF lines results in a loss of pro-tumor immunosuppressive capacities. A. CON CAFs or NetG1 KD CAFs were allowed to produce CM for 48 hours in 3D. The CM was then subjected to ELISA analysis for the quantification of the following cytokines: GM-CSF, IL-6, IL-8, TGF-β, and IL-15. N= 3 biological replicates. One-way ANOVA, Dunnett’s multiple comparison test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay

NetG1 functions are mediated through AKT and p38 pathways. A. Representative western blots demonstrating downregulation of p-AKT and p-p38 in NetG1 KO CAFs compared to CON. GAPDH was used as a loading control. B. RFP + PDACc (2×10 4 ) were co-cultured with CON CAFs pre-treated for 24 hours with DMSO, 10 nM p38 inhibitor (p38i), or 10 µM AKT inhibitor (AKTi) and PDACc survival was assessed 96 hours later. * compared to DMSO. C. Graphs depicting relative Glu and Gln levels measured from CAFs treated with p38i or AKTi in serum/Gln free media for 48 hours. DMSO was used as a treatment control. * Compared to DMSO. D. ELISAs for GM-CSF, IL-6, IL-8, TGF-β, and IL-15 were performed on CM or lysates of CAFs treated for 48 hours with DMSO or p38i ( TOP panel) and DMSO or AKTi ( BOTTOM panel ). * compared to DMSO. Student’s T-test: *p
Figure Legend Snippet: NetG1 functions are mediated through AKT and p38 pathways. A. Representative western blots demonstrating downregulation of p-AKT and p-p38 in NetG1 KO CAFs compared to CON. GAPDH was used as a loading control. B. RFP + PDACc (2×10 4 ) were co-cultured with CON CAFs pre-treated for 24 hours with DMSO, 10 nM p38 inhibitor (p38i), or 10 µM AKT inhibitor (AKTi) and PDACc survival was assessed 96 hours later. * compared to DMSO. C. Graphs depicting relative Glu and Gln levels measured from CAFs treated with p38i or AKTi in serum/Gln free media for 48 hours. DMSO was used as a treatment control. * Compared to DMSO. D. ELISAs for GM-CSF, IL-6, IL-8, TGF-β, and IL-15 were performed on CM or lysates of CAFs treated for 48 hours with DMSO or p38i ( TOP panel) and DMSO or AKTi ( BOTTOM panel ). * compared to DMSO. Student’s T-test: *p

Techniques Used: Western Blot, Cell Culture

3) Product Images from "GM-CSF promoted DC recruitment and survival governs the intestinal mucosal response to enteric attaching-and-effacing bacterial pathogens"

Article Title: GM-CSF promoted DC recruitment and survival governs the intestinal mucosal response to enteric attaching-and-effacing bacterial pathogens

Journal: Cell host & microbe

doi: 10.1016/j.chom.2010.01.006

GM-CSF upregulates epithelial cell expression of the DC chemoattractant CCL22 (A) mRNA transcripts for the three DC chemoattractant chemokines, CCL22, CCL8 and CCL20, were assayed by real time RT-PCR in the colon of B6 and GM-CSF -/- mice left uninfected or infected with C. rodentium (n = 4-5 mice per group at each time point). (B) CCL22 protein was assayed in colon extracts from B6 and GM-CSF -/- mice. Data are mean ± SEM (n = 5 mice per group at each time point). For (A, B) * p
Figure Legend Snippet: GM-CSF upregulates epithelial cell expression of the DC chemoattractant CCL22 (A) mRNA transcripts for the three DC chemoattractant chemokines, CCL22, CCL8 and CCL20, were assayed by real time RT-PCR in the colon of B6 and GM-CSF -/- mice left uninfected or infected with C. rodentium (n = 4-5 mice per group at each time point). (B) CCL22 protein was assayed in colon extracts from B6 and GM-CSF -/- mice. Data are mean ± SEM (n = 5 mice per group at each time point). For (A, B) * p

Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay, Infection

4) Product Images from "Reactivation of Latent Human Cytomegalovirus in CD14+ Monocytes Is Differentiation Dependent"

Article Title: Reactivation of Latent Human Cytomegalovirus in CD14+ Monocytes Is Differentiation Dependent

Journal: Journal of Virology

doi: 10.1128/JVI.75.16.7543-7554.2001

Allo-MDM and ConA-MDM display differences in kinetics and production of cytokines. To compare cytokine production in Allo-MDM and ConA-MDM cultures, PBMC from six histoincompatible donors were stimulated by either allogeneically matching different pairs within the group or stimulating cells from individuals mitogenically. Following adherence of activated PBMC, nonadherent cells were removed from the cultures at 24 h poststimulation, and supernatants were collected from the cultures at the indicated intervals. Parallel cultures of Allo-MDM (red), ConA-MDM (blue), and unstimulated PBMC (green) were established as described in Materials and Methods. Cytokine-specific ELISA analysis was used to determine cytokine expression kinetics in cell-free culture supernatants collected at 6, 12, 24, 36, and 48 h and at 3, 5, and 8 days postisolation. Cytokines analyzed include IFN-γ (A), TNF-α (B), IL-2 (C), IL-3 (D), GM-CSF (E), G-CSF (F), M-CSF (G), IL-10 (H), IL-13 (I), IL-7 (J), TGF-β (K), IL-1β (L), and IL-6 (M).
Figure Legend Snippet: Allo-MDM and ConA-MDM display differences in kinetics and production of cytokines. To compare cytokine production in Allo-MDM and ConA-MDM cultures, PBMC from six histoincompatible donors were stimulated by either allogeneically matching different pairs within the group or stimulating cells from individuals mitogenically. Following adherence of activated PBMC, nonadherent cells were removed from the cultures at 24 h poststimulation, and supernatants were collected from the cultures at the indicated intervals. Parallel cultures of Allo-MDM (red), ConA-MDM (blue), and unstimulated PBMC (green) were established as described in Materials and Methods. Cytokine-specific ELISA analysis was used to determine cytokine expression kinetics in cell-free culture supernatants collected at 6, 12, 24, 36, and 48 h and at 3, 5, and 8 days postisolation. Cytokines analyzed include IFN-γ (A), TNF-α (B), IL-2 (C), IL-3 (D), GM-CSF (E), G-CSF (F), M-CSF (G), IL-10 (H), IL-13 (I), IL-7 (J), TGF-β (K), IL-1β (L), and IL-6 (M).

Techniques Used: Enzyme-linked Immunosorbent Assay, Expressing

Reactivation of latent HCMV in Allo-MDM is dependent on production of IFN-γ. Allo-MDM were established by mixing PBMC from two healthy blood donors ( n = 3 with duplicates), and reactivation of HCMV was detected by expression of the HCMV proteins IE and gB. (A) Confocal microscopy analysis of a representative sample of an Allo-MDM culture which reactivated HCMV. The cells expressed both HCMV IE (red) and gB (green). Magnification, ×630. (B) Reactivation of HCMV was inhibited in Allo-MDM, which were established in the presence of neutralizing antibodies to IFN-γ. In contrast, reactivation of virus was not affected by the addition of neutralizing antibodies directed against IL-1, IL-2, TNF-α, TGF-β, or GM-CSF at the time of establishment of the Allo-MDM cultures. (B) Percent HCMV IE-expressing cells in the Allo-MDM cultures at 52 days poststimulation.
Figure Legend Snippet: Reactivation of latent HCMV in Allo-MDM is dependent on production of IFN-γ. Allo-MDM were established by mixing PBMC from two healthy blood donors ( n = 3 with duplicates), and reactivation of HCMV was detected by expression of the HCMV proteins IE and gB. (A) Confocal microscopy analysis of a representative sample of an Allo-MDM culture which reactivated HCMV. The cells expressed both HCMV IE (red) and gB (green). Magnification, ×630. (B) Reactivation of HCMV was inhibited in Allo-MDM, which were established in the presence of neutralizing antibodies to IFN-γ. In contrast, reactivation of virus was not affected by the addition of neutralizing antibodies directed against IL-1, IL-2, TNF-α, TGF-β, or GM-CSF at the time of establishment of the Allo-MDM cultures. (B) Percent HCMV IE-expressing cells in the Allo-MDM cultures at 52 days poststimulation.

Techniques Used: Expressing, Confocal Microscopy

5) Product Images from "In vitro and in vivo study on the effect of antifungal agents on hematopoietic cells in mice"

Article Title: In vitro and in vivo study on the effect of antifungal agents on hematopoietic cells in mice

Journal: Experimental Biology and Medicine

doi: 10.1177/1535370215590820

Effect of liposomal amphotericin B, voriconazole, and caspofungin in CFU assays of murine BM cells in the presence of the single cytokines SCF (a), IL-3 (b), GM-CSF (c), G-CSF (d), or IL-6 (e). Liposomal amphotericin B in combination with each cytokine significantly increased the number of CFU-GM colonies to 20 ± 8%, 15 ± 3%, 19 ± 4%, 47 ± 7%, or 45 ± 14%, respectively, as compared with the respective controls. Voriconazole plus SCF induced a 30 ± 11% increase with borderline statistical significance, whereas its combination with IL-3, GM-CSF, G-CSF, or IL-6 significantly increased the number of CFU-GM colonies to 16 ± 1%, 17 ± 1%, 45 ± 6%, or 35 ± 7%, respectively, as compared with the respective controls. Caspofungin in combination with SCF, IL-3, GM-CSF, G-CSF, or IL-6 significantly increased the number of CFU-GM colonies to 39 ± 10%, 21 ± 3%, 21 ± 5%, 49 ± 6%, or 55 ± 13%, respectively, as compared with these cytokines alone. n = 4 independent experiments performed in duplicate (mean ± SEM). Paired Student’s t test (*, P
Figure Legend Snippet: Effect of liposomal amphotericin B, voriconazole, and caspofungin in CFU assays of murine BM cells in the presence of the single cytokines SCF (a), IL-3 (b), GM-CSF (c), G-CSF (d), or IL-6 (e). Liposomal amphotericin B in combination with each cytokine significantly increased the number of CFU-GM colonies to 20 ± 8%, 15 ± 3%, 19 ± 4%, 47 ± 7%, or 45 ± 14%, respectively, as compared with the respective controls. Voriconazole plus SCF induced a 30 ± 11% increase with borderline statistical significance, whereas its combination with IL-3, GM-CSF, G-CSF, or IL-6 significantly increased the number of CFU-GM colonies to 16 ± 1%, 17 ± 1%, 45 ± 6%, or 35 ± 7%, respectively, as compared with the respective controls. Caspofungin in combination with SCF, IL-3, GM-CSF, G-CSF, or IL-6 significantly increased the number of CFU-GM colonies to 39 ± 10%, 21 ± 3%, 21 ± 5%, 49 ± 6%, or 55 ± 13%, respectively, as compared with these cytokines alone. n = 4 independent experiments performed in duplicate (mean ± SEM). Paired Student’s t test (*, P

Techniques Used:

6) Product Images from "C3a is required for ILC2 function in allergic airway inflammation"

Article Title: C3a is required for ILC2 function in allergic airway inflammation

Journal: Mucosal immunology

doi: 10.1038/s41385-018-0064-x

C3a regulates ILC-T cell crosstalk. 1×10 4 Flow-sorted lung Lin - (CD11b, CD11c, Gr1, B220, CD19, TCRb, TCRgd, CD49b, CD4, CD8, FcER1)ICOS + IL-33R + ILC2 from wildtype and C3ar1 −/− mice were co-cultured with 1×10 4 CFSE-labelled OVA-transgenic (D011.10Tg) lymph node CD4 + T cells in the presence of 1 ug/ml OVA 323–339 peptide for 5 days. ( a ) Representative flow plot showing pre- and post-sort purity of sorted lung ILC2. ( b ) CFSE-labeled DO11.10Tg T cells proliferation. ( c ) Total T cell numbers (CD3 + CD4 + ), ( d ) IL-2 production, ( e ) frequency IL-13 + CD4 + cells, and supernatant levels of ( f ) IL-13 ( g ) GM-CSF and ( h ) IFNγ production. Data is means+SEM, representative of two independent experiments. All flow cytometry gating is based on FMO controls and, non-CFSE-labeled T cells. *p
Figure Legend Snippet: C3a regulates ILC-T cell crosstalk. 1×10 4 Flow-sorted lung Lin - (CD11b, CD11c, Gr1, B220, CD19, TCRb, TCRgd, CD49b, CD4, CD8, FcER1)ICOS + IL-33R + ILC2 from wildtype and C3ar1 −/− mice were co-cultured with 1×10 4 CFSE-labelled OVA-transgenic (D011.10Tg) lymph node CD4 + T cells in the presence of 1 ug/ml OVA 323–339 peptide for 5 days. ( a ) Representative flow plot showing pre- and post-sort purity of sorted lung ILC2. ( b ) CFSE-labeled DO11.10Tg T cells proliferation. ( c ) Total T cell numbers (CD3 + CD4 + ), ( d ) IL-2 production, ( e ) frequency IL-13 + CD4 + cells, and supernatant levels of ( f ) IL-13 ( g ) GM-CSF and ( h ) IFNγ production. Data is means+SEM, representative of two independent experiments. All flow cytometry gating is based on FMO controls and, non-CFSE-labeled T cells. *p

Techniques Used: Flow Cytometry, Mouse Assay, Cell Culture, Transgenic Assay, Labeling, Cytometry

7) Product Images from "Human Cytomegalovirus Inhibits Differentiation of Monocytes into Dendritic Cells with the Consequence of Depressed Immunological Functions"

Article Title: Human Cytomegalovirus Inhibits Differentiation of Monocytes into Dendritic Cells with the Consequence of Depressed Immunological Functions

Journal: Journal of Virology

doi: 10.1128/JVI.77.20.10943-10956.2003

HCMV infection inhibits cytokine-induced differentiation of monocytes into dendritic cells. (A) HCMV was tested for its ability to inhibit differentiation of monocytes into dendritic cells obtained by IL-4 and GM-CSF stimulation by infecting monocytes with the laboratory strain AD169 or Towne or a clinical isolate (PO) at an MOI of 5. The figure presents a representative sample of a flow cytometric analysis of the expression of the cell surface molecules CD1a ( y axes represent the mean fluorescent intensity of CD1a) and CD14 ( x axes represent the mean fluorescent intensity of CD14) on mock-infected dendritic cells and HCMV-infected monocytes. (B) HCMV was tested for its ability to affect cellular differentiation by infecting cells with the laboratory strain AD169 at an MOI of 0.1 to 5, laboratory strain Towne at an MOI of 5, and a clinical isolate (PO) at an MOI of 5. Supernatants from mock-infected fibroblasts (HL-sup) were also used for infection. (C) To test whether the HCMV-induced block in cellular differentiation was dependent on soluble factors, filtered virus-free supernatants were used for infection of cells. To test if the HCMV-induced block in cellular differentiation was directly dependent on the virus particle, ultracentrifuged pure virus particles were used for infection. To test if the inhibition was dependent upon active virus replication or virus uptake by the cell, we used UV-inactivated (replication-deficient) virus for infection of cells. (D) To determine if the inability of the cells to differentiate into dendritic cells was an HCMV-specific phenomenon, monocytes were infected with measles virus or treated with phorbol myristate acetate and LPS. The percentage of CD1a-positive cells in the individual sample compared to the uninfected control was determined. The data represent mean and standard error of the mean values for six separate experiments.
Figure Legend Snippet: HCMV infection inhibits cytokine-induced differentiation of monocytes into dendritic cells. (A) HCMV was tested for its ability to inhibit differentiation of monocytes into dendritic cells obtained by IL-4 and GM-CSF stimulation by infecting monocytes with the laboratory strain AD169 or Towne or a clinical isolate (PO) at an MOI of 5. The figure presents a representative sample of a flow cytometric analysis of the expression of the cell surface molecules CD1a ( y axes represent the mean fluorescent intensity of CD1a) and CD14 ( x axes represent the mean fluorescent intensity of CD14) on mock-infected dendritic cells and HCMV-infected monocytes. (B) HCMV was tested for its ability to affect cellular differentiation by infecting cells with the laboratory strain AD169 at an MOI of 0.1 to 5, laboratory strain Towne at an MOI of 5, and a clinical isolate (PO) at an MOI of 5. Supernatants from mock-infected fibroblasts (HL-sup) were also used for infection. (C) To test whether the HCMV-induced block in cellular differentiation was dependent on soluble factors, filtered virus-free supernatants were used for infection of cells. To test if the HCMV-induced block in cellular differentiation was directly dependent on the virus particle, ultracentrifuged pure virus particles were used for infection. To test if the inhibition was dependent upon active virus replication or virus uptake by the cell, we used UV-inactivated (replication-deficient) virus for infection of cells. (D) To determine if the inability of the cells to differentiate into dendritic cells was an HCMV-specific phenomenon, monocytes were infected with measles virus or treated with phorbol myristate acetate and LPS. The percentage of CD1a-positive cells in the individual sample compared to the uninfected control was determined. The data represent mean and standard error of the mean values for six separate experiments.

Techniques Used: Infection, Flow Cytometry, Expressing, Cell Differentiation, Blocking Assay, Inhibition

8) Product Images from "TNFSF14 (LIGHT) Exhibits Inflammatory Activities in Lung Fibroblasts Complementary to IL-13 and TGF-β"

Article Title: TNFSF14 (LIGHT) Exhibits Inflammatory Activities in Lung Fibroblasts Complementary to IL-13 and TGF-β

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00576

Homologous to Lymphotoxin, exhibits Inducible expression and competes with HSV Glycoprotein D for binding to HVEM, a receptor expressed on T lymphocytes (LIGHT) upregulates the expression of a select group of chemokines, cytokines, and proteinases in lung fibroblasts. Human lung fibroblasts (HLFs) were left unstimulated (gray bars) or stimulated for 48 h with 50 ng/ml soluble recombinant LIGHT (black bars). (A–E) mRNA fold increase of the indicated molecules. (E) Induction of mRNA for CCL5, GM-CSF, and MMP-9 in HLF with siRNA knockdown of LTβR or HVEM. All data are mean ± SEM from triplicate cultures from three independent experiments, shown as fold increase above unstimulated. (F,G) Mean IL-6 (F) and GM-CSF (G) protein levels in HLF supernatants after 3 days. n = 5 independent experiments.
Figure Legend Snippet: Homologous to Lymphotoxin, exhibits Inducible expression and competes with HSV Glycoprotein D for binding to HVEM, a receptor expressed on T lymphocytes (LIGHT) upregulates the expression of a select group of chemokines, cytokines, and proteinases in lung fibroblasts. Human lung fibroblasts (HLFs) were left unstimulated (gray bars) or stimulated for 48 h with 50 ng/ml soluble recombinant LIGHT (black bars). (A–E) mRNA fold increase of the indicated molecules. (E) Induction of mRNA for CCL5, GM-CSF, and MMP-9 in HLF with siRNA knockdown of LTβR or HVEM. All data are mean ± SEM from triplicate cultures from three independent experiments, shown as fold increase above unstimulated. (F,G) Mean IL-6 (F) and GM-CSF (G) protein levels in HLF supernatants after 3 days. n = 5 independent experiments.

Techniques Used: Expressing, Binding Assay, Recombinant

9) Product Images from "Netrin G1 promotes pancreatic tumorigenesis through cancer associated fibroblast driven nutritional support and immunosuppression"

Article Title: Netrin G1 promotes pancreatic tumorigenesis through cancer associated fibroblast driven nutritional support and immunosuppression

Journal: bioRxiv

doi: 10.1101/330209

NetG1 + CAFs create an immunosuppressive microenvironment that protects PDAC cells from NK cell induced death. A. Quantification of U-Plex (multiplex ELISA; GM-CSF, IL-1β, CCL20, IL-6, IL-8) and ELISAs (IL-15, TGF-β) of assorted cytokines with immunomodulatory or immunoattractive potentials, detected in the CM of TA, CON CAFs, or NetG1 KO CAFs, growing in 3D. N= 6 biological replicates. * compared to TA. B. Quantification of the % of NK-92 cells positive for markers of activation (IFNγ and Granzyme B) determined by flow cytometry after IL-2 pre-activated NK-92 cells (8×10 4 ) were in direct co-culture (CC) with CON or NetG1 KO CAFs (2×10 4 ) or treated with their conditioned media (CM) for 16 hours. * compared to CON CAF CC. C. Primary NK cells (10 5 ) were isolated from healthy human donors, pre-activated with IL-2/IL-12, incubated with CM from CON or NetG1 KO CAFs for 16 hours, and their activation status was determined by flow cytometry, using IFNγ and CD69 as markers. Expression of markers was normalized to the positive control (IL-2 alone = 1.0). * comparison between the CON and KO at each % of CM. D-E. RFP + CON or NGL-1 KO PDACc (2×10 4 ) were co-cultured in 3D with GFP + CON or NetG1 KO CAFs (2×10 4 ) and with active (D) or resting (E) NK-92 cells (8×10 4 ) for 48 hours and PDAC survival was quantified. Groups were normalized to CON PDACc/CON CAF with active NK cells. Dotted line in the resting graph (RIGHT) denotes PDACc survival with CON PDACc/CON CAF with active NK cells. * compared to CON PDACc/CON CAF with active NK cells. One-Way ANOVA, Dunnett’s multiple comparison test (A, B, D) or T-test (C). *p
Figure Legend Snippet: NetG1 + CAFs create an immunosuppressive microenvironment that protects PDAC cells from NK cell induced death. A. Quantification of U-Plex (multiplex ELISA; GM-CSF, IL-1β, CCL20, IL-6, IL-8) and ELISAs (IL-15, TGF-β) of assorted cytokines with immunomodulatory or immunoattractive potentials, detected in the CM of TA, CON CAFs, or NetG1 KO CAFs, growing in 3D. N= 6 biological replicates. * compared to TA. B. Quantification of the % of NK-92 cells positive for markers of activation (IFNγ and Granzyme B) determined by flow cytometry after IL-2 pre-activated NK-92 cells (8×10 4 ) were in direct co-culture (CC) with CON or NetG1 KO CAFs (2×10 4 ) or treated with their conditioned media (CM) for 16 hours. * compared to CON CAF CC. C. Primary NK cells (10 5 ) were isolated from healthy human donors, pre-activated with IL-2/IL-12, incubated with CM from CON or NetG1 KO CAFs for 16 hours, and their activation status was determined by flow cytometry, using IFNγ and CD69 as markers. Expression of markers was normalized to the positive control (IL-2 alone = 1.0). * comparison between the CON and KO at each % of CM. D-E. RFP + CON or NGL-1 KO PDACc (2×10 4 ) were co-cultured in 3D with GFP + CON or NetG1 KO CAFs (2×10 4 ) and with active (D) or resting (E) NK-92 cells (8×10 4 ) for 48 hours and PDAC survival was quantified. Groups were normalized to CON PDACc/CON CAF with active NK cells. Dotted line in the resting graph (RIGHT) denotes PDACc survival with CON PDACc/CON CAF with active NK cells. * compared to CON PDACc/CON CAF with active NK cells. One-Way ANOVA, Dunnett’s multiple comparison test (A, B, D) or T-test (C). *p

Techniques Used: Multiplex Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Flow Cytometry, Co-Culture Assay, Isolation, Incubation, Expressing, Positive Control, Cell Culture

Knockdown of NetG1 by CRISPRi in two patient CAF lines results in a loss of pro-tumor immunosuppressive capacities. A. CON CAFs or NetG1 KD CAFs were allowed to produce CM for 48 hours in 3D. The CM was then subjected to ELISA analysis for the quantification of the following cytokines: GM-CSF, IL-6, IL-8, TGF-β, and IL-15. N= 3 biological replicates. One-way ANOVA, Dunnett’s multiple comparison test, * p
Figure Legend Snippet: Knockdown of NetG1 by CRISPRi in two patient CAF lines results in a loss of pro-tumor immunosuppressive capacities. A. CON CAFs or NetG1 KD CAFs were allowed to produce CM for 48 hours in 3D. The CM was then subjected to ELISA analysis for the quantification of the following cytokines: GM-CSF, IL-6, IL-8, TGF-β, and IL-15. N= 3 biological replicates. One-way ANOVA, Dunnett’s multiple comparison test, * p

Techniques Used: Enzyme-linked Immunosorbent Assay

NetG1 functions are mediated through AKT and p38 pathways. A. Representative western blots demonstrating downregulation of p-AKT and p-p38 in NetG1 KO CAFs compared to CON. GAPDH was used as a loading control. B. RFP + PDACc (2×10 4 ) were co-cultured with CON CAFs pre-treated for 24 hours with DMSO, 10 nM p38 inhibitor (p38i), or 10 µM AKT inhibitor (AKTi) and PDACc survival was assessed 96 hours later. * compared to DMSO. C. Graphs depicting relative Glu and Gln levels measured from CAFs treated with p38i or AKTi in serum/Gln free media for 48 hours. DMSO was used as a treatment control. * Compared to DMSO. D. ELISAs for GM-CSF, IL-6, IL-8, TGF-β, and IL-15 were performed on CM or lysates of CAFs treated for 48 hours with DMSO or p38i ( TOP panel) and DMSO or AKTi ( BOTTOM panel ). * compared to DMSO. Student’s T-test: *p
Figure Legend Snippet: NetG1 functions are mediated through AKT and p38 pathways. A. Representative western blots demonstrating downregulation of p-AKT and p-p38 in NetG1 KO CAFs compared to CON. GAPDH was used as a loading control. B. RFP + PDACc (2×10 4 ) were co-cultured with CON CAFs pre-treated for 24 hours with DMSO, 10 nM p38 inhibitor (p38i), or 10 µM AKT inhibitor (AKTi) and PDACc survival was assessed 96 hours later. * compared to DMSO. C. Graphs depicting relative Glu and Gln levels measured from CAFs treated with p38i or AKTi in serum/Gln free media for 48 hours. DMSO was used as a treatment control. * Compared to DMSO. D. ELISAs for GM-CSF, IL-6, IL-8, TGF-β, and IL-15 were performed on CM or lysates of CAFs treated for 48 hours with DMSO or p38i ( TOP panel) and DMSO or AKTi ( BOTTOM panel ). * compared to DMSO. Student’s T-test: *p

Techniques Used: Western Blot, Cell Culture

10) Product Images from "Anti-Granulocyte-Macrophage Colony-Stimulating Factor Autoantibodies Are a Risk Factor for Central Nervous System Infection by Cryptococcus gattii in Otherwise Immunocompetent Patients"

Article Title: Anti-Granulocyte-Macrophage Colony-Stimulating Factor Autoantibodies Are a Risk Factor for Central Nervous System Infection by Cryptococcus gattii in Otherwise Immunocompetent Patients

Journal: mBio

doi: 10.1128/mBio.00912-14

Anti-GM-CSF autoantibodies containing plasma blunts the response of PBMCs to GM-CSF stimulation. (A) A representative dose-response curve was depicted by measuring p-STAT5 production in normal PBMCs with 10% plasma from a patient or from a healthy individual under conditions of stimulation at increasing concentrations (between 0.001 ng/ml and 10 µg/ml) of GM-CSF. The concentration of GM-CSF required to phosphorylate 50% of STAT5 (EC 50 ) was 2,298 ng/ml ( R 2 = 0.9998) and 0.6609 ng/ml ( R 2 = 0.9990) for the patient’s plasma and for the normal plasma, respectively. (B) The concentration of GM-CSF required to phosphorylate 50% of STAT5 (EC 50 ) was determined from the dose-response curves generated for each of the anti-GM-CSF autoantibody-positive CNS cryptococcosis (CNSC) patients’ plasma samples ( n = 7), plasma from anti-GM-CSF autoantibody-negative healthy volunteers ( n = 5), and plasma from an anti-GM-CSF autoantibody-positive healthy volunteer ( n = 1). Aab, anti-GM-CSF autoantibodies.
Figure Legend Snippet: Anti-GM-CSF autoantibodies containing plasma blunts the response of PBMCs to GM-CSF stimulation. (A) A representative dose-response curve was depicted by measuring p-STAT5 production in normal PBMCs with 10% plasma from a patient or from a healthy individual under conditions of stimulation at increasing concentrations (between 0.001 ng/ml and 10 µg/ml) of GM-CSF. The concentration of GM-CSF required to phosphorylate 50% of STAT5 (EC 50 ) was 2,298 ng/ml ( R 2 = 0.9998) and 0.6609 ng/ml ( R 2 = 0.9990) for the patient’s plasma and for the normal plasma, respectively. (B) The concentration of GM-CSF required to phosphorylate 50% of STAT5 (EC 50 ) was determined from the dose-response curves generated for each of the anti-GM-CSF autoantibody-positive CNS cryptococcosis (CNSC) patients’ plasma samples ( n = 7), plasma from anti-GM-CSF autoantibody-negative healthy volunteers ( n = 5), and plasma from an anti-GM-CSF autoantibody-positive healthy volunteer ( n = 1). Aab, anti-GM-CSF autoantibodies.

Techniques Used: Concentration Assay, Generated

Inhibitory function of anti-GM-CSF autoantibodies. (A) Normal peripheral blood mononuclear cells (PBMCs) were incubated with 10% plasma of each anti-GM-CSF autoantibody-positive CNS cryptococcosis (CNSC) patient ( n = 7) or of each anti-GM-CSF autoantibody-negative healthy volunteer ( n = 5) in the presence or absence of GM-CSF (10 ng/ml) for 30 min and production of p-STAT5 was evaluated by flow cytometry. The stimulation index (ratio of stimulated/unstimulated geometric mean channels) was calculated. aab, autoantibodies. (B) Evaluation of the inhibition of p-STAT5 production by the plasma from one healthy control in China who was positive for anti-GM-CSF autoantibodies. Normal PBMCs were left unstimulated or were stimulated by 10 ng/ml·GM-CSF (cerebrospinal fluid) in the presence of one representative healthy plasma sample negative for anti-GM-CSF autoantibodies (Healthy/Aab−), of one healthy plasma sample positive for anti-GM-CSF autoantibodies (Healthy/Aab+), or of one representative CNS cryptococcosis patient’s plasma sample positive for anti-GM-CSF autoantibodies (CNSC/Aab+), and p-STAT5 production was measured by flow cytometry by intracellular staining for p-STAT5.
Figure Legend Snippet: Inhibitory function of anti-GM-CSF autoantibodies. (A) Normal peripheral blood mononuclear cells (PBMCs) were incubated with 10% plasma of each anti-GM-CSF autoantibody-positive CNS cryptococcosis (CNSC) patient ( n = 7) or of each anti-GM-CSF autoantibody-negative healthy volunteer ( n = 5) in the presence or absence of GM-CSF (10 ng/ml) for 30 min and production of p-STAT5 was evaluated by flow cytometry. The stimulation index (ratio of stimulated/unstimulated geometric mean channels) was calculated. aab, autoantibodies. (B) Evaluation of the inhibition of p-STAT5 production by the plasma from one healthy control in China who was positive for anti-GM-CSF autoantibodies. Normal PBMCs were left unstimulated or were stimulated by 10 ng/ml·GM-CSF (cerebrospinal fluid) in the presence of one representative healthy plasma sample negative for anti-GM-CSF autoantibodies (Healthy/Aab−), of one healthy plasma sample positive for anti-GM-CSF autoantibodies (Healthy/Aab+), or of one representative CNS cryptococcosis patient’s plasma sample positive for anti-GM-CSF autoantibodies (CNSC/Aab+), and p-STAT5 production was measured by flow cytometry by intracellular staining for p-STAT5.

Techniques Used: Incubation, Flow Cytometry, Cytometry, Inhibition, Staining

11) Product Images from "CLEC5A Mediates Macrophage Function and COPD Pathologies"

Article Title: CLEC5A Mediates Macrophage Function and COPD Pathologies

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

doi: 10.4049/jimmunol.1500978

CLEC5A and LPS or GM-CSF co-stimulate macrophage from CS-exposed mice BAL macrophage (5×10 4 ) were stimulated with ( A ) control IgG, α-CLEC5A activating antibody (10 ug/ml), LPS (10 ng/ml), or both for 24hrs. Similarly, BAL macrophage (5×10 4 ) were stimulated with ( B ) control IgG, α-CLEC5A activating antibody (10 ug/ml), GM-CSF (20 ng/ml), or both for 24hrs. TNFα was measured by ELISA. Data are mean +/− sem of 3 experiments performed in duplicate. * p
Figure Legend Snippet: CLEC5A and LPS or GM-CSF co-stimulate macrophage from CS-exposed mice BAL macrophage (5×10 4 ) were stimulated with ( A ) control IgG, α-CLEC5A activating antibody (10 ug/ml), LPS (10 ng/ml), or both for 24hrs. Similarly, BAL macrophage (5×10 4 ) were stimulated with ( B ) control IgG, α-CLEC5A activating antibody (10 ug/ml), GM-CSF (20 ng/ml), or both for 24hrs. TNFα was measured by ELISA. Data are mean +/− sem of 3 experiments performed in duplicate. * p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

12) Product Images from "Heparin-Coated Albumin Nanoparticles for Drug Combination in Targeting Inflamed Intestine"

Article Title: Heparin-Coated Albumin Nanoparticles for Drug Combination in Targeting Inflamed Intestine

Journal: Advanced healthcare materials

doi: 10.1002/adhm.202000536

Synthesis and characterization of heparin-coated human serum albumin nanoparticles (HEP-HSA NPs). ( a ) Schematic outlining the formation of drug-loaded HEP-HSA NPs. ( b ) A scanning electron microscopic image of HEP-HSA NPs. ( c, d ) Size, polydispersity index (PDI), and zeta potential measurement of NPs prepared from 40, 80, and 120 mg mL −1 of HSA, before and after HEP coating. ( e ) Coating efficiency (%) of 1.0 mg mL −1 HEP in water on different HSA NPs. ( f, g ) Size, PDI, and zeta potential of HSA NPs loaded with different drugs, before and after HEP coating. BUD: budesonide; Vanco: vancomycin; GM-CSF: granulocyte macrophage colony-stimulating factor. ( h ) Encapsulation efficiency (EE) and the amount of drug loaded in HSA NPs. ( i, j ) Size, PDI, and zeta potential of unloaded and dual-drug loaded HSA NPs with BUD (3 mg mL −1 ) and GM-CSF (50 and 100 μg mL −1 , respectively), before and after HEP coating. ( k ) EE and the amount of BUD and GM-CSF in the dual-drug loaded HSA NPs. Data are presented as mean ± SD from a representative experiment (n = 3; independent experiment was performed at least twice).
Figure Legend Snippet: Synthesis and characterization of heparin-coated human serum albumin nanoparticles (HEP-HSA NPs). ( a ) Schematic outlining the formation of drug-loaded HEP-HSA NPs. ( b ) A scanning electron microscopic image of HEP-HSA NPs. ( c, d ) Size, polydispersity index (PDI), and zeta potential measurement of NPs prepared from 40, 80, and 120 mg mL −1 of HSA, before and after HEP coating. ( e ) Coating efficiency (%) of 1.0 mg mL −1 HEP in water on different HSA NPs. ( f, g ) Size, PDI, and zeta potential of HSA NPs loaded with different drugs, before and after HEP coating. BUD: budesonide; Vanco: vancomycin; GM-CSF: granulocyte macrophage colony-stimulating factor. ( h ) Encapsulation efficiency (EE) and the amount of drug loaded in HSA NPs. ( i, j ) Size, PDI, and zeta potential of unloaded and dual-drug loaded HSA NPs with BUD (3 mg mL −1 ) and GM-CSF (50 and 100 μg mL −1 , respectively), before and after HEP coating. ( k ) EE and the amount of BUD and GM-CSF in the dual-drug loaded HSA NPs. Data are presented as mean ± SD from a representative experiment (n = 3; independent experiment was performed at least twice).

Techniques Used:

13) Product Images from "Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M"

Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

Journal: Nature Communications

doi: 10.1038/ncomms7062

DEX synergistically enhances the NTHi-induced IRAK-M expression in vitro and in vivo . ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P
Figure Legend Snippet: DEX synergistically enhances the NTHi-induced IRAK-M expression in vitro and in vivo . ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P

Techniques Used: Expressing, In Vitro, In Vivo, Mouse Assay, Real-time Polymerase Chain Reaction, Immunostaining, Staining

14) Product Images from "Fetal exposure to HIV-1 alters chemokine receptor expression by CD4+T cells and increases susceptibility to HIV-1"

Article Title: Fetal exposure to HIV-1 alters chemokine receptor expression by CD4+T cells and increases susceptibility to HIV-1

Journal: Scientific Reports

doi: 10.1038/srep06690

Hierarchical clustering of the expression of the chemokine receptors on CD4 + T cells at baseline and after stimulation. The heatmap shows the hierarchical clustering of the expression levels of the chemokine receptors on CD4 + T cells at baseline and after culture with various stimuli, measured by flow cytometry. The - e- after chemoreceptor indicates HIV-1-exposed infants and the -c- healthy controls. CCR7, CXCR4 grouped together, as well as chemokine receptor-related inflammatory responses, i.e. CCR5, CCR8 and CXCR6. The expression of inflammatory response-related chemokine receptors such as CCR5, CCR8 and CXCR6 increased in in vitro cultures in particular with IL-1β and GM-CSF, clustering these two cytokines together. N = 7 in both groups.
Figure Legend Snippet: Hierarchical clustering of the expression of the chemokine receptors on CD4 + T cells at baseline and after stimulation. The heatmap shows the hierarchical clustering of the expression levels of the chemokine receptors on CD4 + T cells at baseline and after culture with various stimuli, measured by flow cytometry. The - e- after chemoreceptor indicates HIV-1-exposed infants and the -c- healthy controls. CCR7, CXCR4 grouped together, as well as chemokine receptor-related inflammatory responses, i.e. CCR5, CCR8 and CXCR6. The expression of inflammatory response-related chemokine receptors such as CCR5, CCR8 and CXCR6 increased in in vitro cultures in particular with IL-1β and GM-CSF, clustering these two cytokines together. N = 7 in both groups.

Techniques Used: Expressing, Flow Cytometry, Cytometry, In Vitro

Chemokine receptor expression on CD4 + T cells after in vitro culture analysed with flow cytometry. Mononuclear cell fractions derived from cord blood of either healthy controls or HIV-1-exposed infants were labelled with CFSE and cultured for 6 days with various stimuli: IL-2, IL-4, IL-7, IL-15, IL-1β GM-CSF and T cell receptor (TCR)-mediated activation with monoclonal antibodies against CD3 and CD28. Expression of chemokine receptors and CFSE was measured by flow cytometry. CD4 + T cells are shown of a HIV-1-exposed infant after stimulation. Stimulation with monoclonal antibodies against CD3 and CD28 induced proliferation in almost all CD4 + T cells ( > 99%), measured by dilution of CFSE. Culture with IL-1β or GM-CSF induced the highest upregulation of CCR5 and CCR8. Proliferating cells in all cultures except in cultures with antibodies against CD3 and CD28 downregulated CCR7 expression.
Figure Legend Snippet: Chemokine receptor expression on CD4 + T cells after in vitro culture analysed with flow cytometry. Mononuclear cell fractions derived from cord blood of either healthy controls or HIV-1-exposed infants were labelled with CFSE and cultured for 6 days with various stimuli: IL-2, IL-4, IL-7, IL-15, IL-1β GM-CSF and T cell receptor (TCR)-mediated activation with monoclonal antibodies against CD3 and CD28. Expression of chemokine receptors and CFSE was measured by flow cytometry. CD4 + T cells are shown of a HIV-1-exposed infant after stimulation. Stimulation with monoclonal antibodies against CD3 and CD28 induced proliferation in almost all CD4 + T cells ( > 99%), measured by dilution of CFSE. Culture with IL-1β or GM-CSF induced the highest upregulation of CCR5 and CCR8. Proliferating cells in all cultures except in cultures with antibodies against CD3 and CD28 downregulated CCR7 expression.

Techniques Used: Expressing, In Vitro, Flow Cytometry, Cytometry, Derivative Assay, Cell Culture, Activation Assay

15) Product Images from "Neural Stem Cells Engineered to Express Three Therapeutic Factors Mediate Recovery from Chronic Stage CNS Autoimmunity"

Article Title: Neural Stem Cells Engineered to Express Three Therapeutic Factors Mediate Recovery from Chronic Stage CNS Autoimmunity

Journal: Molecular Therapy

doi: 10.1038/mt.2016.104

Effect of cocktail-NSCs on the acute stage of EAE. ( a ) EAE mice were i.v. injected with various NSCs (1 × 10 6 cells/mouse) at onset of disease (day 10 p.i.) and then i.p. injected with Dox every two days ( n = 5 each group). Mice were scored blindly for disease severity daily by two researchers according to a 0–5 scale. ( b ) Accumulative score of EAE (sum of daily clinical scores from day 11 to day 28 p.i.). ( c ) Mice were sacrificed at day 28 p.i. ( n = 5 each group), and spinal cords were harvested for H E stained spinal cord sections. The white matter of the lumbar spinal cord was analyzed to assess inflammation. ( d ) Mean score of inflammation in H E staining. ( e–g ) Spinal cords and brains were harvested and MNCs isolated ( n = 5 each group). ( e ) Total MNC numbers in the CNS were counted under light microscopy. ( f ) Percentages of CD4 + , CD8 + , CD11b + , and CD11c + cells and ( g ) IFN- γ + , IL-17 + , and GM-CSF + CD4 + T cells were determined by flow cytometry. Absolute numbers of different subtypes of CNS infiltrating cells were calculated by multiplying the percentages of these cells (as shown in Supplementary Figure S4 ) by total numbers of MNCs obtained from each spinal cord and brain tissue. Groups designated by the same letter are not significantly different, while those with different letters (A, B, C, D, E, or F) are significantly different ( P
Figure Legend Snippet: Effect of cocktail-NSCs on the acute stage of EAE. ( a ) EAE mice were i.v. injected with various NSCs (1 × 10 6 cells/mouse) at onset of disease (day 10 p.i.) and then i.p. injected with Dox every two days ( n = 5 each group). Mice were scored blindly for disease severity daily by two researchers according to a 0–5 scale. ( b ) Accumulative score of EAE (sum of daily clinical scores from day 11 to day 28 p.i.). ( c ) Mice were sacrificed at day 28 p.i. ( n = 5 each group), and spinal cords were harvested for H E stained spinal cord sections. The white matter of the lumbar spinal cord was analyzed to assess inflammation. ( d ) Mean score of inflammation in H E staining. ( e–g ) Spinal cords and brains were harvested and MNCs isolated ( n = 5 each group). ( e ) Total MNC numbers in the CNS were counted under light microscopy. ( f ) Percentages of CD4 + , CD8 + , CD11b + , and CD11c + cells and ( g ) IFN- γ + , IL-17 + , and GM-CSF + CD4 + T cells were determined by flow cytometry. Absolute numbers of different subtypes of CNS infiltrating cells were calculated by multiplying the percentages of these cells (as shown in Supplementary Figure S4 ) by total numbers of MNCs obtained from each spinal cord and brain tissue. Groups designated by the same letter are not significantly different, while those with different letters (A, B, C, D, E, or F) are significantly different ( P

Techniques Used: Mouse Assay, Injection, Staining, Isolation, Light Microscopy, Flow Cytometry, Cytometry

16) Product Images from "Pharmacokinetics of combined gene therapy expressing constitutive human GM-CSF and hyperthermia-regulated human IL-12"

Article Title: Pharmacokinetics of combined gene therapy expressing constitutive human GM-CSF and hyperthermia-regulated human IL-12

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/1756-9966-32-5

hGM-CSF and hIL-12 expression in heat treated A549 and Hep3B cells. A549 and Hep3B cells in 24-well plates were infected with Adcmv-hGMCSF-hsp-hIL12 virus for 24 hrs and heated at 45°C for 45 min. Twenty-four hours late, medium was collected for hGM-CSF and hIL-12 measurement. A ) hIL-12 expression under heating and no heating treatment. B ) hGM-CSF expression under heating and no heating treatment. C ) Relative hGM-CSF and hIL-12 expression in A549 cells. D ) Relative hGM-CSF and hIL-12 expression in Hep3B cells. HT: heating treatment. N = 5 repeated experiments.
Figure Legend Snippet: hGM-CSF and hIL-12 expression in heat treated A549 and Hep3B cells. A549 and Hep3B cells in 24-well plates were infected with Adcmv-hGMCSF-hsp-hIL12 virus for 24 hrs and heated at 45°C for 45 min. Twenty-four hours late, medium was collected for hGM-CSF and hIL-12 measurement. A ) hIL-12 expression under heating and no heating treatment. B ) hGM-CSF expression under heating and no heating treatment. C ) Relative hGM-CSF and hIL-12 expression in A549 cells. D ) Relative hGM-CSF and hIL-12 expression in Hep3B cells. HT: heating treatment. N = 5 repeated experiments.

Techniques Used: Expressing, Infection

The time dependence of hGM-CSF and hIL-12 expression in heat treated A549 and Hep3B cells. Cells were infected and heated as described in Figure 2 . Medium was collected at 24 and 48 hrs after heating treatment. A ) hIL-12 expression in A549 and Hep3B cells. B ) hGM-CSF expression in A549 and Hep3b cells. C ) Comparison of hIL-12 expression between cells heated for 24 hrs and cells without heating for 24 and 48 hrs. D ) Comparison of hGM-CSF expression between cells heated for 24 hrs and cells without heating for 24 and 48 hrs. N = 5 repeated experiments.
Figure Legend Snippet: The time dependence of hGM-CSF and hIL-12 expression in heat treated A549 and Hep3B cells. Cells were infected and heated as described in Figure 2 . Medium was collected at 24 and 48 hrs after heating treatment. A ) hIL-12 expression in A549 and Hep3B cells. B ) hGM-CSF expression in A549 and Hep3b cells. C ) Comparison of hIL-12 expression between cells heated for 24 hrs and cells without heating for 24 and 48 hrs. D ) Comparison of hGM-CSF expression between cells heated for 24 hrs and cells without heating for 24 and 48 hrs. N = 5 repeated experiments.

Techniques Used: Expressing, Infection

hGM-CSF and hIL-12 expression in Hep3B tumor tissues. Adcmv-GMCSF-hsp-hIL12 was intratumorly injected. Tumors were not heated, heated for 1 time, 2 time, and 3 times at 42°C for 40 min. Animals were sacrificed at different time point and tumor tissues were homogenized for hGM-CSF and hIL12 detection. A ) hIL-12 expression in tumor tissues. B ) hGM-CSF expression in tumor tissues. N = 5 mice per group.
Figure Legend Snippet: hGM-CSF and hIL-12 expression in Hep3B tumor tissues. Adcmv-GMCSF-hsp-hIL12 was intratumorly injected. Tumors were not heated, heated for 1 time, 2 time, and 3 times at 42°C for 40 min. Animals were sacrificed at different time point and tumor tissues were homogenized for hGM-CSF and hIL12 detection. A ) hIL-12 expression in tumor tissues. B ) hGM-CSF expression in tumor tissues. N = 5 mice per group.

Techniques Used: Expressing, Injection, Mouse Assay

17) Product Images from "Comparison of morphology, phenotypes and function between cultured human IL-4-DC and IFN-DC"

Article Title: Comparison of morphology, phenotypes and function between cultured human IL-4-DC and IFN-DC

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2017.7581

Morphology of mIFN-DC and mIL-4-DC derived from CD14 + monocytes. Mature DCs were produced in vitro by culturing monocytes with IFN-α and GM-CSF or GM-CSF and IL-4. Then TNF-α was used to promote the maturation. The scanning electron micoscopy and FACS analysis were conducted to compare the morphologies of these two DCs. (A and B) Scanning electron micoscopy photographs of the integral and the local of mIFN-DC and mIL-4-DC, respectively. (C) FACS analysis of mIFN-DC, mIL-4-DC and monocytes. Nonviable cells were eliminated from analysis. (D) Forward scatter values, generated by FACS, revealed the sizes of mIFN-DC, mIL-4-DC and monocytes. Results were representative of 5 independent experiments. Statistical analysis comparing the size of different DCs and monocytes was performed with the independent-sample t-test (**P
Figure Legend Snippet: Morphology of mIFN-DC and mIL-4-DC derived from CD14 + monocytes. Mature DCs were produced in vitro by culturing monocytes with IFN-α and GM-CSF or GM-CSF and IL-4. Then TNF-α was used to promote the maturation. The scanning electron micoscopy and FACS analysis were conducted to compare the morphologies of these two DCs. (A and B) Scanning electron micoscopy photographs of the integral and the local of mIFN-DC and mIL-4-DC, respectively. (C) FACS analysis of mIFN-DC, mIL-4-DC and monocytes. Nonviable cells were eliminated from analysis. (D) Forward scatter values, generated by FACS, revealed the sizes of mIFN-DC, mIL-4-DC and monocytes. Results were representative of 5 independent experiments. Statistical analysis comparing the size of different DCs and monocytes was performed with the independent-sample t-test (**P

Techniques Used: Derivative Assay, Produced, In Vitro, FACS, Generated

18) Product Images from "Influenza induces IL-8 and GM-CSF secretion by human alveolar epithelial cells through HGF/c-Met and TGF-α/EGFR signaling"

Article Title: Influenza induces IL-8 and GM-CSF secretion by human alveolar epithelial cells through HGF/c-Met and TGF-α/EGFR signaling

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

doi: 10.1152/ajplung.00290.2014

rhHGF and rhTGF-α stimulate IL-8 and GM-CSF production of alveolar type II (ATII) cells, but the basal levels of IL-8 and the level of IL-8 and GM-CSF stimulated by PR8 are not reduced by the EGFR inhibitor. A : concentration of IL-8 in the culture medium of ATII cells with or without rhHGF or rhTGF-α and with or without PR8 infection (MOI = 0.5) and with or without AG1478, an EGFR inhibitor, for 24 h was measured by ELISA. This is the representative data from 3 independent experiments for IL-8. DMSO was a vehicle control for AG1478. B : GM-CSF concentration of culture medium of ATII cells with or without rhHGF or rhTGF-α and with or without PR8 infection (MOI = 0.5) and with or without 10 μM AG1478, an EGFR inhibitor, for 24 h was measured by ELISA. This is the representative data from 3 independent experiments for GM-CSF. DMSO was a vehicle control for AG1478.
Figure Legend Snippet: rhHGF and rhTGF-α stimulate IL-8 and GM-CSF production of alveolar type II (ATII) cells, but the basal levels of IL-8 and the level of IL-8 and GM-CSF stimulated by PR8 are not reduced by the EGFR inhibitor. A : concentration of IL-8 in the culture medium of ATII cells with or without rhHGF or rhTGF-α and with or without PR8 infection (MOI = 0.5) and with or without AG1478, an EGFR inhibitor, for 24 h was measured by ELISA. This is the representative data from 3 independent experiments for IL-8. DMSO was a vehicle control for AG1478. B : GM-CSF concentration of culture medium of ATII cells with or without rhHGF or rhTGF-α and with or without PR8 infection (MOI = 0.5) and with or without 10 μM AG1478, an EGFR inhibitor, for 24 h was measured by ELISA. This is the representative data from 3 independent experiments for GM-CSF. DMSO was a vehicle control for AG1478.

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

Recombinant human hepatocyte growth factor (rhHGF) and rh transforming growth factor (TGF)-α induce human alveolar epithelial cells (AECs) to secrete IL-8 and granulocyte macrophage colony-stimulating factor (GM-CSF) strongly, and IL-6 and monocyte chemotactic protein-1 (MCP-1) moderately, which attract neutrophils. A : IL-8, GM-CSF, IL-6, and MCP-1 protein concentrations in culture medium of human AECs stimulated by 50 ng/ml rhHGF or 10 ng/ml rhTGF-α for 24 h were measured by MILLPLEX; n = 6. Open bar, no growth factor; solid bar, rhHGF; shaded bar, rhTGF-α. The range of maximal stimulation: IL-8, 4,792.3–20,136.3 pg/ml; GM-CSF, 58.6–295.5 pg/ml; IL-6, 11.6–497.5 pg/ml; MCP-1, 26,184.5–104,165.1 pg/ml. B : degree of neutrophil migration was measured in vitro using conditioned medium AECs stimulated by rhTGF-α or rhHGF ±2.5 μM AG1478, ±500 nM PHA665752; n = 3. 1 μM N -Formyl-Met-Leu-Phe (fMLP), 10 ng/ml rhIL-8, and 10 ng/ml rhGM-CSF are positive controls. PMN, polymorphonuclear leukocytes. C : correlation diagram between IL-8 protein concentration in the same samples as B and the degree of neutrophil migration ( y = −883.184 + 0.670 * x ; R 2 = 0.831) * P
Figure Legend Snippet: Recombinant human hepatocyte growth factor (rhHGF) and rh transforming growth factor (TGF)-α induce human alveolar epithelial cells (AECs) to secrete IL-8 and granulocyte macrophage colony-stimulating factor (GM-CSF) strongly, and IL-6 and monocyte chemotactic protein-1 (MCP-1) moderately, which attract neutrophils. A : IL-8, GM-CSF, IL-6, and MCP-1 protein concentrations in culture medium of human AECs stimulated by 50 ng/ml rhHGF or 10 ng/ml rhTGF-α for 24 h were measured by MILLPLEX; n = 6. Open bar, no growth factor; solid bar, rhHGF; shaded bar, rhTGF-α. The range of maximal stimulation: IL-8, 4,792.3–20,136.3 pg/ml; GM-CSF, 58.6–295.5 pg/ml; IL-6, 11.6–497.5 pg/ml; MCP-1, 26,184.5–104,165.1 pg/ml. B : degree of neutrophil migration was measured in vitro using conditioned medium AECs stimulated by rhTGF-α or rhHGF ±2.5 μM AG1478, ±500 nM PHA665752; n = 3. 1 μM N -Formyl-Met-Leu-Phe (fMLP), 10 ng/ml rhIL-8, and 10 ng/ml rhGM-CSF are positive controls. PMN, polymorphonuclear leukocytes. C : correlation diagram between IL-8 protein concentration in the same samples as B and the degree of neutrophil migration ( y = −883.184 + 0.670 * x ; R 2 = 0.831) * P

Techniques Used: Recombinant, Migration, In Vitro, Protein Concentration

Influenza virus infection of AECs induces TGF-α secretion and activates EGFR, which stimulates secretion of IL-8 and GM-CSF by AECs. A : TGF-α concentration of culture medium from AECs with or without influenza A virus/Puerto Rico/8/1934 (PR8) infection (multiplicity of infection, MOI = 0.5) for 24 h was measured by ELISA. Values were means ± SE for 5 different human donors. * P
Figure Legend Snippet: Influenza virus infection of AECs induces TGF-α secretion and activates EGFR, which stimulates secretion of IL-8 and GM-CSF by AECs. A : TGF-α concentration of culture medium from AECs with or without influenza A virus/Puerto Rico/8/1934 (PR8) infection (multiplicity of infection, MOI = 0.5) for 24 h was measured by ELISA. Values were means ± SE for 5 different human donors. * P

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

HGF/c-Met signaling induces IL-8 and GM-CSF secretion by AECs cocultured with lung fibroblasts (FBs) during influenza infection. AECs plated on the inserts were preincubated by PHA665752, a c-Met inhibitor, or vehicle control at 2 h before infection and then infected with PR8 (MOI = 0.5). At 1 h after infection, the AECs were placed over lung FBs plated on the plates to start coculture, and the culture medium was harvested at 48 h after starting the coculture. A : IL-8 concentration of these culture medium was measured by ELISA ( n = 3). DMSO was a vehicle control for PHA665752. The range of maximal stimulation: IL-8, 4,188.1–26,022.0 pg/ml. B : GM-CSF concentration of same culture medium as A was measured by ELISA ( n = 3). DMSO was a vehicle control for PHA665752. The range of maximal stimulation: GM-CSF, 44.0–953.5 pg/ml.
Figure Legend Snippet: HGF/c-Met signaling induces IL-8 and GM-CSF secretion by AECs cocultured with lung fibroblasts (FBs) during influenza infection. AECs plated on the inserts were preincubated by PHA665752, a c-Met inhibitor, or vehicle control at 2 h before infection and then infected with PR8 (MOI = 0.5). At 1 h after infection, the AECs were placed over lung FBs plated on the plates to start coculture, and the culture medium was harvested at 48 h after starting the coculture. A : IL-8 concentration of these culture medium was measured by ELISA ( n = 3). DMSO was a vehicle control for PHA665752. The range of maximal stimulation: IL-8, 4,188.1–26,022.0 pg/ml. B : GM-CSF concentration of same culture medium as A was measured by ELISA ( n = 3). DMSO was a vehicle control for PHA665752. The range of maximal stimulation: GM-CSF, 44.0–953.5 pg/ml.

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

19) Product Images from "Signal Transducer and Activator of Transcription 3 (Stat3C) Promotes Myeloid-Derived Suppressor Cell Expansion and Immune Suppression during Lung Tumorigenesis"

Article Title: Signal Transducer and Activator of Transcription 3 (Stat3C) Promotes Myeloid-Derived Suppressor Cell Expansion and Immune Suppression during Lung Tumorigenesis

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2011.06.028

Concentrations of MDSC-stimulating cytokines in BALF and plasma of CCSP-rtTA/(tetO) 7 -CMV-Stat3C bitransgenic mice. A: The concentrations of IL-1β, IL-6, IL-10, TNF-α, G-CSF, M-CSF, and GM-CSF in BALF of lung from bitransgenic mice that
Figure Legend Snippet: Concentrations of MDSC-stimulating cytokines in BALF and plasma of CCSP-rtTA/(tetO) 7 -CMV-Stat3C bitransgenic mice. A: The concentrations of IL-1β, IL-6, IL-10, TNF-α, G-CSF, M-CSF, and GM-CSF in BALF of lung from bitransgenic mice that

Techniques Used: Mouse Assay

20) Product Images from "Blockage of Eosinopoiesis by IL-17A Is Prevented by Cytokine and Lipid Mediators of Allergic Inflammation"

Article Title: Blockage of Eosinopoiesis by IL-17A Is Prevented by Cytokine and Lipid Mediators of Allergic Inflammation

Journal: Mediators of Inflammation

doi: 10.1155/2015/968932

IL-17, effect on progenitor responses and requirement for IL-17RA. (a, b) Semisolid cultures were established for 7 days, from 2 × 10 5 bone-marrow cells of naive BALB/c mice, in the presence of GM-CSF (2 ng/mL), alone or in association with IL-17A at the indicated concentrations. Total (a) and differential (b) colony counts were carried out under the inverted microscope, directly (a) or after staining dried agar layers for EPO and counterstaining by Harris' Hematoxylin (b). Colony types in (b) are [ 36 ] pure granulocyte-macrophage (GM), mixed granulocyte-macrophage-eosinophil (GMEos), pure eosinophil (Eos), pure granulocyte (G), and pure macrophage (M). GM-CSF alone, black bar. GM-CSF in association with IL-17A (0.1 to 1 ng/mL), white bars. (c) Liquid cultures were established as described by Gaspar-Elsas et al. [ 38 ], in a two-phase culture with progenitor expansion in Flt3L and SCF, followed by eosinophil differentiation for up to 10 days with GM-CSF, IL-3, and IL-5, in the absence (white bar) or presence (black bar) of IL-17A (1 ng/mL). (d) Liquid cultures were established as in Figure 1(a) , with bone-marrow from BALB/c mutants lacking IL-17RA. Data are mean ± SEM. ∗ P
Figure Legend Snippet: IL-17, effect on progenitor responses and requirement for IL-17RA. (a, b) Semisolid cultures were established for 7 days, from 2 × 10 5 bone-marrow cells of naive BALB/c mice, in the presence of GM-CSF (2 ng/mL), alone or in association with IL-17A at the indicated concentrations. Total (a) and differential (b) colony counts were carried out under the inverted microscope, directly (a) or after staining dried agar layers for EPO and counterstaining by Harris' Hematoxylin (b). Colony types in (b) are [ 36 ] pure granulocyte-macrophage (GM), mixed granulocyte-macrophage-eosinophil (GMEos), pure eosinophil (Eos), pure granulocyte (G), and pure macrophage (M). GM-CSF alone, black bar. GM-CSF in association with IL-17A (0.1 to 1 ng/mL), white bars. (c) Liquid cultures were established as described by Gaspar-Elsas et al. [ 38 ], in a two-phase culture with progenitor expansion in Flt3L and SCF, followed by eosinophil differentiation for up to 10 days with GM-CSF, IL-3, and IL-5, in the absence (white bar) or presence (black bar) of IL-17A (1 ng/mL). (d) Liquid cultures were established as in Figure 1(a) , with bone-marrow from BALB/c mutants lacking IL-17RA. Data are mean ± SEM. ∗ P

Techniques Used: Mouse Assay, Inverted Microscopy, Staining

21) Product Images from "A neuropeptide, Substance-P, directly induces tissue-repairing M2 like macrophages by activating the PI3K/Akt/mTOR pathway even in the presence of IFNγ"

Article Title: A neuropeptide, Substance-P, directly induces tissue-repairing M2 like macrophages by activating the PI3K/Akt/mTOR pathway even in the presence of IFNγ

Journal: Scientific Reports

doi: 10.1038/s41598-017-09639-7

SP induces M2 polarization through early induction of Arginase-1. ( a ) Mo BM were collected after 1-h of attachment and differentiated into MΦ GM-CSF by GM-CSF treatment for 5 d. MΦ GM-CSF (CD14 − CD11b + CD68 + CCR7 + CD206 + ) were defined as M1-like precursors that were differentiated from Mo BM (CD14 + CD11b + CD68 − CCR7 − CD206 + ). For the immunofluorescence staining results (Supplementary Fig. 1a ), the number of positive-cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm 2 ) (n = 3). ( b ) CD14 + MACS-isolated monocytes were differentiated with GM-CSF treatment for 5 d, and cells were further characterized as CD11b + CD68 + macrophages. (Green = CD11b, Red = CD68, Blue = DAPI) (n = 3). ( c ) Proposed model for MΦ GM-CSF from Mo BM with 80% CD14 + and 20% CD14 − phenotypes. ( d ) Increased Arginase-1 activity was detected at the indicated time point of SP treatment using the Arginase-1 activity assay, and the results were compared with typical M1/M2-inducing cytokines (n = 3). ( e ) The increased expression of Arginase-1 was detected after 6 h of SP treatment. The western blot samples derived from the same experiment and were quantified with ImageJ. Full-length blots are presented in Supplementary Figure 16a (n = 4). ( f – g ) Dose-dependent increased activity and expression of Arginase-1 determined after 6 h of SP treatment. This was completely blocked by pretreatment with the NK-1R antagonist RP67580 (1 μM). ( f ) Arginase-1 activity assay (n = 3). ( g ) The western blot samples derived from the same experiment and were quantified with ImageJ. Full-length blots are presented in Supplementary Figure 16b (n = 4). ( h ) Increased NO was detected in culture with IFNγ, GM-CSF, or LPS but not with SP, IL-4/13, or IL-10. Quantitative analysis of NO production was performed in conditioned medium from MΦ GM-CSF that had been treated with SP or cytokines for 1 d. LPS treatment served as a positive control (n = 6). Data represent the mean ± SEM (* p
Figure Legend Snippet: SP induces M2 polarization through early induction of Arginase-1. ( a ) Mo BM were collected after 1-h of attachment and differentiated into MΦ GM-CSF by GM-CSF treatment for 5 d. MΦ GM-CSF (CD14 − CD11b + CD68 + CCR7 + CD206 + ) were defined as M1-like precursors that were differentiated from Mo BM (CD14 + CD11b + CD68 − CCR7 − CD206 + ). For the immunofluorescence staining results (Supplementary Fig. 1a ), the number of positive-cells was counted (5 random fields/coverslip) at 100 × magnification (0.85 mm 2 ) (n = 3). ( b ) CD14 + MACS-isolated monocytes were differentiated with GM-CSF treatment for 5 d, and cells were further characterized as CD11b + CD68 + macrophages. (Green = CD11b, Red = CD68, Blue = DAPI) (n = 3). ( c ) Proposed model for MΦ GM-CSF from Mo BM with 80% CD14 + and 20% CD14 − phenotypes. ( d ) Increased Arginase-1 activity was detected at the indicated time point of SP treatment using the Arginase-1 activity assay, and the results were compared with typical M1/M2-inducing cytokines (n = 3). ( e ) The increased expression of Arginase-1 was detected after 6 h of SP treatment. The western blot samples derived from the same experiment and were quantified with ImageJ. Full-length blots are presented in Supplementary Figure 16a (n = 4). ( f – g ) Dose-dependent increased activity and expression of Arginase-1 determined after 6 h of SP treatment. This was completely blocked by pretreatment with the NK-1R antagonist RP67580 (1 μM). ( f ) Arginase-1 activity assay (n = 3). ( g ) The western blot samples derived from the same experiment and were quantified with ImageJ. Full-length blots are presented in Supplementary Figure 16b (n = 4). ( h ) Increased NO was detected in culture with IFNγ, GM-CSF, or LPS but not with SP, IL-4/13, or IL-10. Quantitative analysis of NO production was performed in conditioned medium from MΦ GM-CSF that had been treated with SP or cytokines for 1 d. LPS treatment served as a positive control (n = 6). Data represent the mean ± SEM (* p

Techniques Used: Immunofluorescence, Staining, Magnetic Cell Separation, Isolation, Activity Assay, Expressing, Western Blot, Derivative Assay, Positive Control

22) Product Images from "FGL2 promotes tumor progression in the CNS by suppressing CD103+ dendritic cell differentiation"

Article Title: FGL2 promotes tumor progression in the CNS by suppressing CD103+ dendritic cell differentiation

Journal: Nature Communications

doi: 10.1038/s41467-018-08271-x

FGL2 suppresses GM-CSF-dependent CD103 induction on cDCs in vitro. a Expression of CD103 on CD11c + B220 − DCs cultured with conditioned medium (CM) for 5 or 15 days. Bone marrow cells were isolated from C57BL/6 mice and then cultured with Ctrl -CM (CM from Ctrl tumor cells) or FGL2KO -CM (CM from FGL2KO tumor cells) for 5 days or 15 days. Non-adherent cells were recovered, stained, and analyzed by FACS to determine the expression levels of CD103, Clec9A, and MHCII on the CD11c + B220 − DCs. b OT-I T cell activation by OVA-pulsed CM-cultured bone marrow DCs. Non-adherent bone marrow DCs were recovered from CM-cultured bone marrow DCs at 15 days. The bone marrow DCs (2 × 10 4 ) were incubated with soluble OVA protein (100 μg/mL) for 2 h and then washed twice before co-culturing with the CFSE-labeled OT-I cells (5 × 10 4 ). OT-I activation was measured by CD69 expression level, which was determined by flow cytometry. c Protein levels of GM-CSF and FLT3L in medium conditioned by tumor cells were detected by ELISA. Data were summarized from five independent experiments and were analyzed by paired t -test. d Representative FACS assay of CD103 expression on CD11c + B220 − DCs cultured with FGL2KO-CM in the presence of neutralization antibody anti-GM-CSF (5 μg/mL) or anti-FLT3L (5 μg/mL) for 5 days. Data were summarized from four independent experiments and were analyzed by paired t -test. ** P
Figure Legend Snippet: FGL2 suppresses GM-CSF-dependent CD103 induction on cDCs in vitro. a Expression of CD103 on CD11c + B220 − DCs cultured with conditioned medium (CM) for 5 or 15 days. Bone marrow cells were isolated from C57BL/6 mice and then cultured with Ctrl -CM (CM from Ctrl tumor cells) or FGL2KO -CM (CM from FGL2KO tumor cells) for 5 days or 15 days. Non-adherent cells were recovered, stained, and analyzed by FACS to determine the expression levels of CD103, Clec9A, and MHCII on the CD11c + B220 − DCs. b OT-I T cell activation by OVA-pulsed CM-cultured bone marrow DCs. Non-adherent bone marrow DCs were recovered from CM-cultured bone marrow DCs at 15 days. The bone marrow DCs (2 × 10 4 ) were incubated with soluble OVA protein (100 μg/mL) for 2 h and then washed twice before co-culturing with the CFSE-labeled OT-I cells (5 × 10 4 ). OT-I activation was measured by CD69 expression level, which was determined by flow cytometry. c Protein levels of GM-CSF and FLT3L in medium conditioned by tumor cells were detected by ELISA. Data were summarized from five independent experiments and were analyzed by paired t -test. d Representative FACS assay of CD103 expression on CD11c + B220 − DCs cultured with FGL2KO-CM in the presence of neutralization antibody anti-GM-CSF (5 μg/mL) or anti-FLT3L (5 μg/mL) for 5 days. Data were summarized from four independent experiments and were analyzed by paired t -test. ** P

Techniques Used: In Vitro, Expressing, Cell Culture, Isolation, Mouse Assay, Staining, FACS, Activation Assay, Incubation, Labeling, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Neutralization

23) Product Images from "Pharmacologic inhibition of protein phosphatase-2A achieves durable immune-mediated antitumor activity when combined with PD-1 blockade"

Article Title: Pharmacologic inhibition of protein phosphatase-2A achieves durable immune-mediated antitumor activity when combined with PD-1 blockade

Journal: Nature Communications

doi: 10.1038/s41467-018-04425-z

PP2A inhibition enhances T-cell function in human mixed lymphocyte reactions. a DCs were induced from purified monocytes by culturing in IL4 and GM-CSF for 7 days. 10 5 purified CFSE labeled CD4+ T cells were then co-cultured with 10 4 allogenic DCs in the presence of LB-100 titration in duplicates or triplicates for 5 days. LB-100 was replenished on day 3. Supernatant was collected on day 5 and measured for IFN-γ production. FACS analysis was performed on the cultured cells. b In vitro proliferation of CD4+ T cells in the presence of LB-100 dose titration, measured by dilution of the cytosolic CFSE. Percentage of cells divided was plotted against concentration of LB-100 and representative flow cytometry data demonstrated increase in % cells divided at 1 μM of LB-100. c IFN-γ production measured at day 5 demonstrated a dose-dependent increase in IFN-γ secretion with LB-100. d Intracellular staining of T-bet was performed in CD4+ T cells after 5 days of co-culture. Percentage of CD4+ T-bet+ (of CD4+ cells) was increased at 1 μM of LB-100 . e IFN-γ production in cells treated with isotype control, LB-100 and/or Nivolumab demonstrated a synergistic increase in IFN-γ production with combination treatment. 1 μM of LB-100 and 0.5 nM (75 ng ml −1 ) of Nivolumab, * P
Figure Legend Snippet: PP2A inhibition enhances T-cell function in human mixed lymphocyte reactions. a DCs were induced from purified monocytes by culturing in IL4 and GM-CSF for 7 days. 10 5 purified CFSE labeled CD4+ T cells were then co-cultured with 10 4 allogenic DCs in the presence of LB-100 titration in duplicates or triplicates for 5 days. LB-100 was replenished on day 3. Supernatant was collected on day 5 and measured for IFN-γ production. FACS analysis was performed on the cultured cells. b In vitro proliferation of CD4+ T cells in the presence of LB-100 dose titration, measured by dilution of the cytosolic CFSE. Percentage of cells divided was plotted against concentration of LB-100 and representative flow cytometry data demonstrated increase in % cells divided at 1 μM of LB-100. c IFN-γ production measured at day 5 demonstrated a dose-dependent increase in IFN-γ secretion with LB-100. d Intracellular staining of T-bet was performed in CD4+ T cells after 5 days of co-culture. Percentage of CD4+ T-bet+ (of CD4+ cells) was increased at 1 μM of LB-100 . e IFN-γ production in cells treated with isotype control, LB-100 and/or Nivolumab demonstrated a synergistic increase in IFN-γ production with combination treatment. 1 μM of LB-100 and 0.5 nM (75 ng ml −1 ) of Nivolumab, * P

Techniques Used: Inhibition, Cell Function Assay, Purification, Labeling, Cell Culture, Titration, FACS, In Vitro, Concentration Assay, Flow Cytometry, Cytometry, Staining, Co-Culture Assay

24) Product Images from "Tumor-Derived Microvesicles Enhance Cross-Processing Ability of Clinical Grade Dendritic Cells"

Article Title: Tumor-Derived Microvesicles Enhance Cross-Processing Ability of Clinical Grade Dendritic Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02481

Clinical grade DCs display different biological features compared to standard S-DCs . (A) Morphological differences of DCs grown in RPMI + 10%FBS (S-DCs) and in X-VIVO 15 (X-DCs) (a,b, respectively) visualized by phase contrast inverted microscope (ZEISS West Germany IM35, 3,2X). (B) Flow cytometry analysis of immature and mature DCs (iDCs and mDCs, respectively) grown in FBS-RPMI or X-VIVO 15 after 6 day culture in the presence of GM-CSF, IL-4. iDCs were matured with IL-6, IL-1β, PGE-2, and TNF-α on day 5. IgG 1 PE and FITC were used as isotype controls and employed to evaluate fluorescence signal background and set the gate. Results were shown as value of Mean Fluorescence Intensity (MFI) of each phenotypic marker subtracted of the corresponded negative control MFI value and depicted as scatter plot (black circle for S-DCs; black triangles for X-DCs). Statistically significant differences between S-DCs vs X-DCs were indicated (* p
Figure Legend Snippet: Clinical grade DCs display different biological features compared to standard S-DCs . (A) Morphological differences of DCs grown in RPMI + 10%FBS (S-DCs) and in X-VIVO 15 (X-DCs) (a,b, respectively) visualized by phase contrast inverted microscope (ZEISS West Germany IM35, 3,2X). (B) Flow cytometry analysis of immature and mature DCs (iDCs and mDCs, respectively) grown in FBS-RPMI or X-VIVO 15 after 6 day culture in the presence of GM-CSF, IL-4. iDCs were matured with IL-6, IL-1β, PGE-2, and TNF-α on day 5. IgG 1 PE and FITC were used as isotype controls and employed to evaluate fluorescence signal background and set the gate. Results were shown as value of Mean Fluorescence Intensity (MFI) of each phenotypic marker subtracted of the corresponded negative control MFI value and depicted as scatter plot (black circle for S-DCs; black triangles for X-DCs). Statistically significant differences between S-DCs vs X-DCs were indicated (* p

Techniques Used: Inverted Microscopy, Flow Cytometry, Cytometry, Fluorescence, Marker, Negative Control

25) Product Images from "Dendritic Cells-based Vaccine and Immune Monitoring for Hepatocellular Carcinoma"

Article Title: Dendritic Cells-based Vaccine and Immune Monitoring for Hepatocellular Carcinoma

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

doi: 10.4196/kjpp.2010.14.1.11

Cells with long cytoplasmic projections were visible at 12 days of culture in a medium containing GM-CSF, IL-4, TNF-α and FL. Original magnification ×400.
Figure Legend Snippet: Cells with long cytoplasmic projections were visible at 12 days of culture in a medium containing GM-CSF, IL-4, TNF-α and FL. Original magnification ×400.

Techniques Used:

Phase contrast micrograph of freshly isolated peripheral blood stem cells showing a homogenous population of equally sized and round cells. Original magnification ×200 (A). After 3 days of culture with the medium containing GM-CSF, IL-4, TNF-α
Figure Legend Snippet: Phase contrast micrograph of freshly isolated peripheral blood stem cells showing a homogenous population of equally sized and round cells. Original magnification ×200 (A). After 3 days of culture with the medium containing GM-CSF, IL-4, TNF-α

Techniques Used: Isolation

26) Product Images from "PKM2 promotes Th17 cell differentiation and autoimmune inflammation by fine-tuning STAT3 activation"

Article Title: PKM2 promotes Th17 cell differentiation and autoimmune inflammation by fine-tuning STAT3 activation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20190613

Loss of PKM2 in CD4 T cells does not impair Th1, Th2 or iT reg differentiation. (A) WT or PKM2-deficient CD4 T were cultured under Th17 cell–skewing conditions and stained for both IL-17A and Foxp3, followed by flow cytometric analysis ( n = 5). (B–D) Naive CD4 T cells were also cultured under Th1, Th2, or iT reg-skewing conditions and analyzed for expression of IFNγ, IL-4, and Foxp3, respectively, by flow cytometry ( n = 3). In addition, IFN-γ and IL-13 levels in supernatants of Th1 and Th2 cultures, respectively, were measured by ELISA ( n = 3). (E) Naive CD4 T cells were cultured under Th1 or Th17 cell–polarizing conditions for 96 h. Intracellular staining for IFN-γ and GM-CSF in Th1 cells (top) and both IL-17A and GM-CSF in Th17 cells (bottom) was performed, followed by flow cytometric analysis ( n = 5). Data are representative of at least three independent experiments. Error bars show mean ± SEM. P values were determined by two-tailed Student’s t test. *, P
Figure Legend Snippet: Loss of PKM2 in CD4 T cells does not impair Th1, Th2 or iT reg differentiation. (A) WT or PKM2-deficient CD4 T were cultured under Th17 cell–skewing conditions and stained for both IL-17A and Foxp3, followed by flow cytometric analysis ( n = 5). (B–D) Naive CD4 T cells were also cultured under Th1, Th2, or iT reg-skewing conditions and analyzed for expression of IFNγ, IL-4, and Foxp3, respectively, by flow cytometry ( n = 3). In addition, IFN-γ and IL-13 levels in supernatants of Th1 and Th2 cultures, respectively, were measured by ELISA ( n = 3). (E) Naive CD4 T cells were cultured under Th1 or Th17 cell–polarizing conditions for 96 h. Intracellular staining for IFN-γ and GM-CSF in Th1 cells (top) and both IL-17A and GM-CSF in Th17 cells (bottom) was performed, followed by flow cytometric analysis ( n = 5). Data are representative of at least three independent experiments. Error bars show mean ± SEM. P values were determined by two-tailed Student’s t test. *, P

Techniques Used: Cell Culture, Staining, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

PKM2 boosts Th17 cell-mediated EAE pathogenesis. (A) EAE was induced in WT or CD4 Cre Pkm2 fl/fl mice and DLN cells collected on day 15 ( n = 5 per group). Cells were stimulated and intracellularly stained for IL-17A or Foxp3, followed by flow cytometric analysis. (B) DLN cells were harvested and restimulated with MOG 35–55 in vitro for 72 h. The supernatants were collected, and the levels of IL-17A, GM-CSF, and IFN-γ were measured by ELISA ( n = 5). (C) Lumbar spinal cord sections were collected from naive or EAE mice with PKM2 deficiency in CD4 T cells. Homogenates were obtained and mRNA extracted, followed by cDNA conversion; RT-qPCR was performed to analyze the expression of Il17a , Csf2 , and Ifng . Gapdh was used for normalization ( n = 5). (D) DLN cells were collected from WT or CD4 Cre Pkm2 fl/fl EAE mice (day 8) and cultured in the presence of MOG 35-55 under Th17 cell–skewing conditions for 72 h. CD4 T cells were sorted and intravenously transferred (10 6 ) into Rag1 −/− mice. 1 d later, EAE was induced in the recipient mice ( n = 6 per group). Mice were monitored for clinical signs of EAE and CNS inflammatory cell infiltrate analyzed by H E staining. Scale bar represents 50 µm. (E) PKM2 and phospho-PKM2 (Y105) protein levels in the spinal cord of EAE-bearing mice were determined by immunoblot. β-actin was used as a loading control. Data are representative of two (A–D) or three (E) independent experiments. Error bars show mean ± SEM. P values were determined by two-way ANOVA followed by Tukey’s post hoc test (B–D) and two-tailed Student’s t test (A). *, P
Figure Legend Snippet: PKM2 boosts Th17 cell-mediated EAE pathogenesis. (A) EAE was induced in WT or CD4 Cre Pkm2 fl/fl mice and DLN cells collected on day 15 ( n = 5 per group). Cells were stimulated and intracellularly stained for IL-17A or Foxp3, followed by flow cytometric analysis. (B) DLN cells were harvested and restimulated with MOG 35–55 in vitro for 72 h. The supernatants were collected, and the levels of IL-17A, GM-CSF, and IFN-γ were measured by ELISA ( n = 5). (C) Lumbar spinal cord sections were collected from naive or EAE mice with PKM2 deficiency in CD4 T cells. Homogenates were obtained and mRNA extracted, followed by cDNA conversion; RT-qPCR was performed to analyze the expression of Il17a , Csf2 , and Ifng . Gapdh was used for normalization ( n = 5). (D) DLN cells were collected from WT or CD4 Cre Pkm2 fl/fl EAE mice (day 8) and cultured in the presence of MOG 35-55 under Th17 cell–skewing conditions for 72 h. CD4 T cells were sorted and intravenously transferred (10 6 ) into Rag1 −/− mice. 1 d later, EAE was induced in the recipient mice ( n = 6 per group). Mice were monitored for clinical signs of EAE and CNS inflammatory cell infiltrate analyzed by H E staining. Scale bar represents 50 µm. (E) PKM2 and phospho-PKM2 (Y105) protein levels in the spinal cord of EAE-bearing mice were determined by immunoblot. β-actin was used as a loading control. Data are representative of two (A–D) or three (E) independent experiments. Error bars show mean ± SEM. P values were determined by two-way ANOVA followed by Tukey’s post hoc test (B–D) and two-tailed Student’s t test (A). *, P

Techniques Used: Mouse Assay, Staining, In Vitro, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Cell Culture, Two Tailed Test

27) Product Images from "Consequences of hypoxia for the pulmonary alveolar epithelial cell innate immune response1"

Article Title: Consequences of hypoxia for the pulmonary alveolar epithelial cell innate immune response1

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

doi: 10.4049/jimmunol.1701387

Effects of hypoxia expression of GM-CSF, CCL2 and IL-6 by primary murine AEC. Primary murine AEC were cultured for 24h in normoxic (21% oxygen, black bars) or hypoxic (1% oxygen, gray bars) conditions. Constitutive expression of GM-CSF was determined in media alone ( a and b ). Expression of CCL2 and IL-6 was determined in the absence or presence of IL-1ß (10ng/ml; c and d ). mRNA (RT-PCR) is expressed relative to control (normoxia) with the value of the first control sample set at 100. Protein was measured by ELISA in the culture media collected after 24h. Data are representative of 3 independent experiments and expressed as mean ± SEM, n=3. **p
Figure Legend Snippet: Effects of hypoxia expression of GM-CSF, CCL2 and IL-6 by primary murine AEC. Primary murine AEC were cultured for 24h in normoxic (21% oxygen, black bars) or hypoxic (1% oxygen, gray bars) conditions. Constitutive expression of GM-CSF was determined in media alone ( a and b ). Expression of CCL2 and IL-6 was determined in the absence or presence of IL-1ß (10ng/ml; c and d ). mRNA (RT-PCR) is expressed relative to control (normoxia) with the value of the first control sample set at 100. Protein was measured by ELISA in the culture media collected after 24h. Data are representative of 3 independent experiments and expressed as mean ± SEM, n=3. **p

Techniques Used: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

AEC expression of HIF-1α and of HIF-target genes in hypoxia. Whole cell HIF-1α protein levels were measured by ELISA on cell lysates in primary murine AEC after exposure to normoxia (21% oxygen) or hypoxia (1% oxygen) for 24h (Figure 3a). Data shown are mean ± SEM of three independent AEC isolations, with the cells from each isolation were pooled. Translocation of active HIF-1α to the nucleus in MLE15 cells exposed to hypoxia (1% oxygen) was measured as binding of nuclear protein to HIF-1α transcription elements, detected by HFI1α antibody (Figure 3b). Data presented are the mean of two and are representative of two independent experiments. To confirm functional HIF induction in primary murine AEC, mRNA expression of three classic HIF responsive genes (iNOS, HO-1, VEGF) was determined after 24h in normoxia or hypoxia (Figure 3c). The data are expressed relative to normoxic levels with the value of the first control sample set at 100. The experiment shown is representative of 2 independent experiments. AEC were exposed to the HIF stabilizing agent, DMOG (1mM), for 24h and intracellular HIF-1α (Figure 3d pg protein/10 7 cells), secreted VEGF (Figure 3d, pg/ml) and secreted GM-CSF (Figure 3e) protein levels measured. The experiment shown is representative of 2 independent experiments. **p
Figure Legend Snippet: AEC expression of HIF-1α and of HIF-target genes in hypoxia. Whole cell HIF-1α protein levels were measured by ELISA on cell lysates in primary murine AEC after exposure to normoxia (21% oxygen) or hypoxia (1% oxygen) for 24h (Figure 3a). Data shown are mean ± SEM of three independent AEC isolations, with the cells from each isolation were pooled. Translocation of active HIF-1α to the nucleus in MLE15 cells exposed to hypoxia (1% oxygen) was measured as binding of nuclear protein to HIF-1α transcription elements, detected by HFI1α antibody (Figure 3b). Data presented are the mean of two and are representative of two independent experiments. To confirm functional HIF induction in primary murine AEC, mRNA expression of three classic HIF responsive genes (iNOS, HO-1, VEGF) was determined after 24h in normoxia or hypoxia (Figure 3c). The data are expressed relative to normoxic levels with the value of the first control sample set at 100. The experiment shown is representative of 2 independent experiments. AEC were exposed to the HIF stabilizing agent, DMOG (1mM), for 24h and intracellular HIF-1α (Figure 3d pg protein/10 7 cells), secreted VEGF (Figure 3d, pg/ml) and secreted GM-CSF (Figure 3e) protein levels measured. The experiment shown is representative of 2 independent experiments. **p

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Isolation, Translocation Assay, Binding Assay, Functional Assay

28) Product Images from "Mesothelin-specific T cell cytotoxicity against triple negative breast cancer is enhanced by 40s ribosomal protein subunit 3-treated self-differentiated dendritic cells"

Article Title: Mesothelin-specific T cell cytotoxicity against triple negative breast cancer is enhanced by 40s ribosomal protein subunit 3-treated self-differentiated dendritic cells

Journal: Oncology Reports

doi: 10.3892/or.2022.8338

The lentiviral vector schematic maps and immunophenotype of MSLN-SmartDC and RPS3-MSLN-SmartDC. (A) Construction of MSLN-SmartDC lentiviral vector. (B) Expression of MSLN protein in Lenti-X™ 293T transfected with IRFP-SmartDC and MSLN-SmartDC lentiviral vectors. (C) Representative images of monocytes at day 0 and DCs at day 7 of the experiment. Production of (D) GM-CSF, (E) IL-4 and (F) IL-12p70 by monocytes and different DCs. The geometric mean fluorescence intensity of surface markers of DCs in different treatment conditions including (G) CD14, (H) CD40, (I) HLA-DR, (J) CD80, (K) CD83 and (L) CD86 in monocytes and DCs. Original magnification, ×100 and scale bar=50 µm. The results were collected from four independent experiments. ND, not detected. # P
Figure Legend Snippet: The lentiviral vector schematic maps and immunophenotype of MSLN-SmartDC and RPS3-MSLN-SmartDC. (A) Construction of MSLN-SmartDC lentiviral vector. (B) Expression of MSLN protein in Lenti-X™ 293T transfected with IRFP-SmartDC and MSLN-SmartDC lentiviral vectors. (C) Representative images of monocytes at day 0 and DCs at day 7 of the experiment. Production of (D) GM-CSF, (E) IL-4 and (F) IL-12p70 by monocytes and different DCs. The geometric mean fluorescence intensity of surface markers of DCs in different treatment conditions including (G) CD14, (H) CD40, (I) HLA-DR, (J) CD80, (K) CD83 and (L) CD86 in monocytes and DCs. Original magnification, ×100 and scale bar=50 µm. The results were collected from four independent experiments. ND, not detected. # P

Techniques Used: Plasmid Preparation, Expressing, Transfection, Fluorescence

29) Product Images from "Elevated Kallikrein-binding protein in diabetes impairs wound healing through inducing macrophage M1 polarization"

Article Title: Elevated Kallikrein-binding protein in diabetes impairs wound healing through inducing macrophage M1 polarization

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/s12964-019-0376-9

KBP increases the production of M-CSF and MCP-1, which employs the differentiation and migration of monocytes and macrophages. a The plasma level of M-CSF in WT and KBP-TG mice. b The level of M-CSF in the cell supernatant of RAW 264.7 cells. c The mRNA expression of M-CSF in RAW264.7 cells treated with KBP versus that in the Con group. d KBP stimulates the migration of macrophages. Representative images of the Transwell migration assay and a statistical histogram. e The mRNA expression of MCP-1 in the wounds of WT and KBP-TG mice at different time points. f The mRNA expression of MCP-1 in the wounds of DM mice treated with IgG and KBP antibody at different time points. g The plasma level of MCP-1 in WT and KBP-TG mice. h The level of MCP-1 in the cell supernatant of RAW264.7 cells treated with KBP versus that in the Con group. The supernatant of RAW264.7 cells treated with KBP versus that of the Con group. I. The mRNA expression of MCP-1 in RAW264.7 cells treated with KBP versus that in the Con group. Data are presented as the mean ± SD. n = 3; * p
Figure Legend Snippet: KBP increases the production of M-CSF and MCP-1, which employs the differentiation and migration of monocytes and macrophages. a The plasma level of M-CSF in WT and KBP-TG mice. b The level of M-CSF in the cell supernatant of RAW 264.7 cells. c The mRNA expression of M-CSF in RAW264.7 cells treated with KBP versus that in the Con group. d KBP stimulates the migration of macrophages. Representative images of the Transwell migration assay and a statistical histogram. e The mRNA expression of MCP-1 in the wounds of WT and KBP-TG mice at different time points. f The mRNA expression of MCP-1 in the wounds of DM mice treated with IgG and KBP antibody at different time points. g The plasma level of MCP-1 in WT and KBP-TG mice. h The level of MCP-1 in the cell supernatant of RAW264.7 cells treated with KBP versus that in the Con group. The supernatant of RAW264.7 cells treated with KBP versus that of the Con group. I. The mRNA expression of MCP-1 in RAW264.7 cells treated with KBP versus that in the Con group. Data are presented as the mean ± SD. n = 3; * p

Techniques Used: Migration, Mouse Assay, Expressing, Transwell Migration Assay

30) Product Images from "BAFF-secreting neutrophils drive plasma cell responses during emergency granulopoiesis"

Article Title: BAFF-secreting neutrophils drive plasma cell responses during emergency granulopoiesis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20150577

Neutropenia at the time of immunization enhances DC migration and IL-23 production. (A–E) Subsequent IL-17 production enhances LN Cox2 expression that is crucial for late iLN neutrophilia. (A) Analysis of the iLN 24 h after FITC painting. (B) Analysis of intracellular levels of p40 in CD11c + MHC II + DCs 24 h after CFA. (C) LPS-stimulated GM-CSF–derived BMDCs were co-cultured with sorted transgenic 2D2 CD4 + T cells and apoptotic neutrophils for 72 h. (D) LysM-DTA mice treated with 5 µg anti-CXCL1 and anti-CXCL2 i.p. every second day after CFA or treated with 40 µg INDO i.p. on days 4, 6, 8, 10, 12, and 14. iLNs were analyzed at day 14 after CFA. A.U., arbitrary units. (E) LysM-DTA mice treated with 100 µg anti–IL-17 antibody i.p. on days 2, 4, and 6. iLNs were analyzed at day 7 after CFA. Data are representative of two independent experiments. n = 5/group. Results are mean ± SEM. *, P
Figure Legend Snippet: Neutropenia at the time of immunization enhances DC migration and IL-23 production. (A–E) Subsequent IL-17 production enhances LN Cox2 expression that is crucial for late iLN neutrophilia. (A) Analysis of the iLN 24 h after FITC painting. (B) Analysis of intracellular levels of p40 in CD11c + MHC II + DCs 24 h after CFA. (C) LPS-stimulated GM-CSF–derived BMDCs were co-cultured with sorted transgenic 2D2 CD4 + T cells and apoptotic neutrophils for 72 h. (D) LysM-DTA mice treated with 5 µg anti-CXCL1 and anti-CXCL2 i.p. every second day after CFA or treated with 40 µg INDO i.p. on days 4, 6, 8, 10, 12, and 14. iLNs were analyzed at day 14 after CFA. A.U., arbitrary units. (E) LysM-DTA mice treated with 100 µg anti–IL-17 antibody i.p. on days 2, 4, and 6. iLNs were analyzed at day 7 after CFA. Data are representative of two independent experiments. n = 5/group. Results are mean ± SEM. *, P

Techniques Used: Migration, Expressing, Derivative Assay, Cell Culture, Transgenic Assay, Mouse Assay

31) Product Images from "A GM-CSF/IL-33 Pathway Facilitates Allergic Airway Responses to Sub-Threshold House Dust Mite Exposure"

Article Title: A GM-CSF/IL-33 Pathway Facilitates Allergic Airway Responses to Sub-Threshold House Dust Mite Exposure

Journal: PLoS ONE

doi: 10.1371/journal.pone.0088714

GM-CSF-driven IL-33 expression is IL-1α independent. ( A,B ) IL-33 by ELISA from freeze/thaw human A549 and NHBE cells stimulated in vitro for 24 h with or without neutralizing anti-IL-1α Ab. ( C ) IL-33 and ( D ) IL-1α by ELISA from lung homogenates of WT mice at 18 h after in vivo exposure to HDM or rGM-CSF. N = 5 mice/group. * p
Figure Legend Snippet: GM-CSF-driven IL-33 expression is IL-1α independent. ( A,B ) IL-33 by ELISA from freeze/thaw human A549 and NHBE cells stimulated in vitro for 24 h with or without neutralizing anti-IL-1α Ab. ( C ) IL-33 and ( D ) IL-1α by ELISA from lung homogenates of WT mice at 18 h after in vivo exposure to HDM or rGM-CSF. N = 5 mice/group. * p

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, In Vitro, Mouse Assay, In Vivo

Schematic of the proposed model. Exposure to a variety of environmental stimuli can result in GM-CSF production from bronchial epithelial cells (BEC) and alveolar epithelial type II cells (ATII). GM-CSF induces IL-33 production from ATII cells in an IL-1α-independent manner. Then, IL-33 acts locally on CD11b + dendritic cells (DC), which reside in close proximity to epithelial cells, to upregulate OX40L and enhance antigen presentation. Such activated status of the lung immunological environment facilitates allergic sensitization to even sub-clinical amounts of inhaled allergens and, ultimately, results in the development of allergic asthma.
Figure Legend Snippet: Schematic of the proposed model. Exposure to a variety of environmental stimuli can result in GM-CSF production from bronchial epithelial cells (BEC) and alveolar epithelial type II cells (ATII). GM-CSF induces IL-33 production from ATII cells in an IL-1α-independent manner. Then, IL-33 acts locally on CD11b + dendritic cells (DC), which reside in close proximity to epithelial cells, to upregulate OX40L and enhance antigen presentation. Such activated status of the lung immunological environment facilitates allergic sensitization to even sub-clinical amounts of inhaled allergens and, ultimately, results in the development of allergic asthma.

Techniques Used:

32) Product Images from "IL-1R-MyD88 signaling in keratinocyte transformation and carcinogenesis"

Article Title: IL-1R-MyD88 signaling in keratinocyte transformation and carcinogenesis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20101355

Expression of NF-κB–regulated proinflammatory factors in RAS-transformed keratinocytes requires a functional IL-1–MyD88 axis. (A and D) Real-time PCR analyses 3 d after RAS transduction of Cxcl1 , Csf2 , Mmp9 , and Tnf mRNA expression in control and v-ras Ha –transduced keratinocytes (WT and MyD88 −/− [A] or IL-1R −/− [D]). (B and E) CXCL1, GM-CSF, and TNF concentrations were determined by ELISA in culture supernatants from control and v-ras Ha –transduced keratinocytes (WT and MyD88 −/− [B] or IL-1R −/− [E]) 3 d after RAS transduction. Data shown are representative of three independent experiments, and bars represent the mean ± SEM of five replicates. *, P
Figure Legend Snippet: Expression of NF-κB–regulated proinflammatory factors in RAS-transformed keratinocytes requires a functional IL-1–MyD88 axis. (A and D) Real-time PCR analyses 3 d after RAS transduction of Cxcl1 , Csf2 , Mmp9 , and Tnf mRNA expression in control and v-ras Ha –transduced keratinocytes (WT and MyD88 −/− [A] or IL-1R −/− [D]). (B and E) CXCL1, GM-CSF, and TNF concentrations were determined by ELISA in culture supernatants from control and v-ras Ha –transduced keratinocytes (WT and MyD88 −/− [B] or IL-1R −/− [E]) 3 d after RAS transduction. Data shown are representative of three independent experiments, and bars represent the mean ± SEM of five replicates. *, P

Techniques Used: Expressing, Transformation Assay, Functional Assay, Real-time Polymerase Chain Reaction, Transduction, Enzyme-linked Immunosorbent Assay

33) Product Images from "PRT062607 Achieves Complete Inhibition of the Spleen Tyrosine Kinase at Tolerated Exposures Following Oral Dosing in Healthy Volunteers"

Article Title: PRT062607 Achieves Complete Inhibition of the Spleen Tyrosine Kinase at Tolerated Exposures Following Oral Dosing in Healthy Volunteers

Journal: Journal of Clinical Pharmacology

doi: 10.1002/jcph.794

Inhibition of immune‐complex–mediated DC activation in vitro. (A) Differentiation of purified peripheral blood monocytes into imDCs via IL4/GM‐CSF is shown based on CD14 and CD1a expression. (B) Data represent DC activation as measured by surface expression of CD80/86 and MCHII from replicate (n = 5) experiments. Pretreatment of cells with vehicle (Vh), anti‐FcRIIa antibody (4.3) or an isotype control (mIgG), or various concentrations of PRT062607 (μM) prior to stimulation with opsonized sheep red blood cells (opRBC) is indicated. Bars represent mean percentage of vehicle plus standard deviation. (C) Representative CD80/86 expression data from opRBC (SYK‐dependent)‐stimulated cells (top histogram overlay) and LPS (SYK‐independent)‐stimulated cells (bottom histogram overlay). Unstimulated cells are shown (No Stim); 1 μM PRT062607 was used as indicated (+2607).
Figure Legend Snippet: Inhibition of immune‐complex–mediated DC activation in vitro. (A) Differentiation of purified peripheral blood monocytes into imDCs via IL4/GM‐CSF is shown based on CD14 and CD1a expression. (B) Data represent DC activation as measured by surface expression of CD80/86 and MCHII from replicate (n = 5) experiments. Pretreatment of cells with vehicle (Vh), anti‐FcRIIa antibody (4.3) or an isotype control (mIgG), or various concentrations of PRT062607 (μM) prior to stimulation with opsonized sheep red blood cells (opRBC) is indicated. Bars represent mean percentage of vehicle plus standard deviation. (C) Representative CD80/86 expression data from opRBC (SYK‐dependent)‐stimulated cells (top histogram overlay) and LPS (SYK‐independent)‐stimulated cells (bottom histogram overlay). Unstimulated cells are shown (No Stim); 1 μM PRT062607 was used as indicated (+2607).

Techniques Used: Inhibition, Activation Assay, In Vitro, Purification, Expressing, Standard Deviation

34) Product Images from "A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy"

Article Title: A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy

Journal: Nature Communications

doi: 10.1038/s41467-021-21431-w

Specificity of the digital nanopillar SERS platform for FGF-2 cytokine detection. Representative SEM images of pillar array incubated with FGF-2 SERS nanotags in the presence of a , b FGF-2 (1031 aM), c G-CSF (1031 aM), d GM-CSF (1031 aM), e CX3CL1 (1031 aM), and f PBS. The red circles highlight the existence of SERS nanotags. Panel b is the magnified SEM image of the red-highlighted section in a . It is noted that nanofabrication debris on the sidewall of the pillars can also be seen. Data from one independent experiment.
Figure Legend Snippet: Specificity of the digital nanopillar SERS platform for FGF-2 cytokine detection. Representative SEM images of pillar array incubated with FGF-2 SERS nanotags in the presence of a , b FGF-2 (1031 aM), c G-CSF (1031 aM), d GM-CSF (1031 aM), e CX3CL1 (1031 aM), and f PBS. The red circles highlight the existence of SERS nanotags. Panel b is the magnified SEM image of the red-highlighted section in a . It is noted that nanofabrication debris on the sidewall of the pillars can also be seen. Data from one independent experiment.

Techniques Used: Incubation

Sensitivity for the simultaneous detection of four cytokines. Representative confocal SERS images of fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and fractalkine (CX3CL1) with the concentration of a 2.6 aM, b 26 aM, c 260 aM, d 1031 aM. Colour scale bars indicate Raman intensities from 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), 4-mercaptobenzoic acid (MBA), 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (TFMBA), or 2‐mercapto‐4‐methyl‐5‐thiazoleacetic acid (MMTAA). The median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 for 2.6 aM: 3 (1.5–3), 1 (1–2), 2 (1–3), 2 (1–3); 26 aM: 8 (5.5–10), 10 (9–13), 7 (6–10), 8 (6–10); 260 aM: 40 (36–48), 40 (35–52), 39 (35–50), 37 (36–49); and 1031 aM: 79 (61.5–97), 78 (72–87.5), 88 (68.5–97), 79 (64–95), respectively. Data represents one experiment from three independent tests.
Figure Legend Snippet: Sensitivity for the simultaneous detection of four cytokines. Representative confocal SERS images of fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and fractalkine (CX3CL1) with the concentration of a 2.6 aM, b 26 aM, c 260 aM, d 1031 aM. Colour scale bars indicate Raman intensities from 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), 4-mercaptobenzoic acid (MBA), 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (TFMBA), or 2‐mercapto‐4‐methyl‐5‐thiazoleacetic acid (MMTAA). The median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 for 2.6 aM: 3 (1.5–3), 1 (1–2), 2 (1–3), 2 (1–3); 26 aM: 8 (5.5–10), 10 (9–13), 7 (6–10), 8 (6–10); 260 aM: 40 (36–48), 40 (35–52), 39 (35–50), 37 (36–49); and 1031 aM: 79 (61.5–97), 78 (72–87.5), 88 (68.5–97), 79 (64–95), respectively. Data represents one experiment from three independent tests.

Techniques Used: Concentration Assay

Digital nanopillar SERS assay for monitoring melanoma patients during immune checkpoint therapy. For Patient 1 who developed severe irAEs, SERS images for cytokine detection on a day 7, b day 21, c day 42, d cytokine concentration graph for fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fractalkine (CX3CL1). The two shorter horizontal lines denote the interquartile ranges (25th and 75th percentile) and the longer horizontal lines in between denote the median (50th percentile), and e LDA analysis, respectively. For Patient 6 who developed mild irAEs, SERS images for cytokine detection on f day 0, g day 21, h day 42, i four cytokine concentration graph, the two shorter horizontal lines denote the interquartile ranges (25th and 75th percentile) and the longer horizontal lines in between denote the median (50th percentile), and j LDA analysis, respectively. IPI ipilimumab, PEMBRO pembrolizumab; G3 grade 3, G2 grade 2; SD stable disease, PR partial response. For Patient 1, the median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 on day 7: 14 (11–22.5), 23 (21, 29), 12 (7.5–18), 17 (9–25.5); day 21: 30 (19–37.5), 33 (19–41), 26 (17.5–36.5), 29 (21–43); and day 42: 33 (16.5–58.5), 76 (64–128.5), 25 (14–39.5), 48 (26.5–73.5), respectively. For Patient 6, the median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 on day 0: 18 (16–23), 49 (31.5–56), 23 (17.5–28), 20 (14.5–27); day 21: 29 (24–33.5), 53 (46.5–70), 35 (25–46), 22 (19–29.5); and day 42: 13 (8–16.5), 44 (23.5–55.5), 10 (6.5–12.5), 30 (24–34.5), respectively. The data represented three technical replicates obtained from three chips. Nine images were acquired from each chip for cytokine counting. Statistical analysis was based on Kruskal–Wallis test followed by Dunn’s test to correct multiple comparisons (two-sided). Source data are provided in the Source Data file.
Figure Legend Snippet: Digital nanopillar SERS assay for monitoring melanoma patients during immune checkpoint therapy. For Patient 1 who developed severe irAEs, SERS images for cytokine detection on a day 7, b day 21, c day 42, d cytokine concentration graph for fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fractalkine (CX3CL1). The two shorter horizontal lines denote the interquartile ranges (25th and 75th percentile) and the longer horizontal lines in between denote the median (50th percentile), and e LDA analysis, respectively. For Patient 6 who developed mild irAEs, SERS images for cytokine detection on f day 0, g day 21, h day 42, i four cytokine concentration graph, the two shorter horizontal lines denote the interquartile ranges (25th and 75th percentile) and the longer horizontal lines in between denote the median (50th percentile), and j LDA analysis, respectively. IPI ipilimumab, PEMBRO pembrolizumab; G3 grade 3, G2 grade 2; SD stable disease, PR partial response. For Patient 1, the median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 on day 7: 14 (11–22.5), 23 (21, 29), 12 (7.5–18), 17 (9–25.5); day 21: 30 (19–37.5), 33 (19–41), 26 (17.5–36.5), 29 (21–43); and day 42: 33 (16.5–58.5), 76 (64–128.5), 25 (14–39.5), 48 (26.5–73.5), respectively. For Patient 6, the median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 on day 0: 18 (16–23), 49 (31.5–56), 23 (17.5–28), 20 (14.5–27); day 21: 29 (24–33.5), 53 (46.5–70), 35 (25–46), 22 (19–29.5); and day 42: 13 (8–16.5), 44 (23.5–55.5), 10 (6.5–12.5), 30 (24–34.5), respectively. The data represented three technical replicates obtained from three chips. Nine images were acquired from each chip for cytokine counting. Statistical analysis was based on Kruskal–Wallis test followed by Dunn’s test to correct multiple comparisons (two-sided). Source data are provided in the Source Data file.

Techniques Used: Concentration Assay, Chromatin Immunoprecipitation

Specificity of digital nanopillar SERS platform for FGF-2 cytokine detection. Representative confocal SERS images in the presence of a target FGF-2 (1031 aM), and negative controls with non-target controls b G-CSF (1031 aM), c GM-CSF (1031 aM), d CX3CL1 (1031 aM), and e PBS. The median (interquartile range) of active pillars per scanning image for FGF-2, G-CSF, GM-CSF, CX3CL1, and PBS was 72 (63.5–76.75), 1.5 (1.5–2), 2 (1–4), 0.5 (0–1.25), and 1 (1–1.75), respectively. Data from one independent experiment.
Figure Legend Snippet: Specificity of digital nanopillar SERS platform for FGF-2 cytokine detection. Representative confocal SERS images in the presence of a target FGF-2 (1031 aM), and negative controls with non-target controls b G-CSF (1031 aM), c GM-CSF (1031 aM), d CX3CL1 (1031 aM), and e PBS. The median (interquartile range) of active pillars per scanning image for FGF-2, G-CSF, GM-CSF, CX3CL1, and PBS was 72 (63.5–76.75), 1.5 (1.5–2), 2 (1–4), 0.5 (0–1.25), and 1 (1–1.75), respectively. Data from one independent experiment.

Techniques Used:

Specificity of the digital nanopillar SERS platform for FGF-2 cytokine detection. Representative SEM images of pillar array incubated with FGF-2 SERS nanotags in the presence of a , b FGF-2 (1031 aM), c G-CSF (1031 aM), d GM-CSF (1031 aM), e CX3CL1 (1031 aM), and f PBS. The red circles highlight the existence of SERS nanotags. Panel b is the magnified SEM image of the red-highlighted section in a . It is noted that nanofabrication debris on the sidewall of the pillars can also be seen. Data from one independent experiment.
Figure Legend Snippet: Specificity of the digital nanopillar SERS platform for FGF-2 cytokine detection. Representative SEM images of pillar array incubated with FGF-2 SERS nanotags in the presence of a , b FGF-2 (1031 aM), c G-CSF (1031 aM), d GM-CSF (1031 aM), e CX3CL1 (1031 aM), and f PBS. The red circles highlight the existence of SERS nanotags. Panel b is the magnified SEM image of the red-highlighted section in a . It is noted that nanofabrication debris on the sidewall of the pillars can also be seen. Data from one independent experiment.

Techniques Used: Incubation

Sensitivity for the simultaneous detection of four cytokines. Representative confocal SERS images of fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and fractalkine (CX3CL1) with the concentration of a 2.6 aM, b 26 aM, c 260 aM, d 1031 aM. Colour scale bars indicate Raman intensities from 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), 4-mercaptobenzoic acid (MBA), 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (TFMBA), or 2‐mercapto‐4‐methyl‐5‐thiazoleacetic acid (MMTAA). The median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 for 2.6 aM: 3 (1.5–3), 1 (1–2), 2 (1–3), 2 (1–3); 26 aM: 8 (5.5–10), 10 (9–13), 7 (6–10), 8 (6–10); 260 aM: 40 (36–48), 40 (35–52), 39 (35–50), 37 (36–49); and 1031 aM: 79 (61.5–97), 78 (72–87.5), 88 (68.5–97), 79 (64–95), respectively. Data represents one experiment from three independent tests.
Figure Legend Snippet: Sensitivity for the simultaneous detection of four cytokines. Representative confocal SERS images of fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and fractalkine (CX3CL1) with the concentration of a 2.6 aM, b 26 aM, c 260 aM, d 1031 aM. Colour scale bars indicate Raman intensities from 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), 4-mercaptobenzoic acid (MBA), 2,3,5,6-tetrafluoro-4-mercaptobenzoic acid (TFMBA), or 2‐mercapto‐4‐methyl‐5‐thiazoleacetic acid (MMTAA). The median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 for 2.6 aM: 3 (1.5–3), 1 (1–2), 2 (1–3), 2 (1–3); 26 aM: 8 (5.5–10), 10 (9–13), 7 (6–10), 8 (6–10); 260 aM: 40 (36–48), 40 (35–52), 39 (35–50), 37 (36–49); and 1031 aM: 79 (61.5–97), 78 (72–87.5), 88 (68.5–97), 79 (64–95), respectively. Data represents one experiment from three independent tests.

Techniques Used: Concentration Assay

Digital nanopillar SERS assay for monitoring melanoma patients during immune checkpoint therapy. For Patient 1 who developed severe irAEs, SERS images for cytokine detection on a day 7, b day 21, c day 42, d cytokine concentration graph for fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fractalkine (CX3CL1). The two shorter horizontal lines denote the interquartile ranges (25th and 75th percentile) and the longer horizontal lines in between denote the median (50th percentile), and e LDA analysis, respectively. For Patient 6 who developed mild irAEs, SERS images for cytokine detection on f day 0, g day 21, h day 42, i four cytokine concentration graph, the two shorter horizontal lines denote the interquartile ranges (25th and 75th percentile) and the longer horizontal lines in between denote the median (50th percentile), and j LDA analysis, respectively. IPI ipilimumab, PEMBRO pembrolizumab; G3 grade 3, G2 grade 2; SD stable disease, PR partial response. For Patient 1, the median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 on day 7: 14 (11–22.5), 23 (21, 29), 12 (7.5–18), 17 (9–25.5); day 21: 30 (19–37.5), 33 (19–41), 26 (17.5–36.5), 29 (21–43); and day 42: 33 (16.5–58.5), 76 (64–128.5), 25 (14–39.5), 48 (26.5–73.5), respectively. For Patient 6, the median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 on day 0: 18 (16–23), 49 (31.5–56), 23 (17.5–28), 20 (14.5–27); day 21: 29 (24–33.5), 53 (46.5–70), 35 (25–46), 22 (19–29.5); and day 42: 13 (8–16.5), 44 (23.5–55.5), 10 (6.5–12.5), 30 (24–34.5), respectively. The data represented three technical replicates obtained from three chips. Nine images were acquired from each chip for cytokine counting. Statistical analysis was based on Kruskal–Wallis test followed by Dunn’s test to correct multiple comparisons (two-sided). Source data are provided in the Source Data file.
Figure Legend Snippet: Digital nanopillar SERS assay for monitoring melanoma patients during immune checkpoint therapy. For Patient 1 who developed severe irAEs, SERS images for cytokine detection on a day 7, b day 21, c day 42, d cytokine concentration graph for fibroblast growth factor 2 (FGF-2), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fractalkine (CX3CL1). The two shorter horizontal lines denote the interquartile ranges (25th and 75th percentile) and the longer horizontal lines in between denote the median (50th percentile), and e LDA analysis, respectively. For Patient 6 who developed mild irAEs, SERS images for cytokine detection on f day 0, g day 21, h day 42, i four cytokine concentration graph, the two shorter horizontal lines denote the interquartile ranges (25th and 75th percentile) and the longer horizontal lines in between denote the median (50th percentile), and j LDA analysis, respectively. IPI ipilimumab, PEMBRO pembrolizumab; G3 grade 3, G2 grade 2; SD stable disease, PR partial response. For Patient 1, the median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 on day 7: 14 (11–22.5), 23 (21, 29), 12 (7.5–18), 17 (9–25.5); day 21: 30 (19–37.5), 33 (19–41), 26 (17.5–36.5), 29 (21–43); and day 42: 33 (16.5–58.5), 76 (64–128.5), 25 (14–39.5), 48 (26.5–73.5), respectively. For Patient 6, the median (interquartile range) of active pillars per scanning image of FGF-2, G-CSF, GM-CSF, CX3CL1 on day 0: 18 (16–23), 49 (31.5–56), 23 (17.5–28), 20 (14.5–27); day 21: 29 (24–33.5), 53 (46.5–70), 35 (25–46), 22 (19–29.5); and day 42: 13 (8–16.5), 44 (23.5–55.5), 10 (6.5–12.5), 30 (24–34.5), respectively. The data represented three technical replicates obtained from three chips. Nine images were acquired from each chip for cytokine counting. Statistical analysis was based on Kruskal–Wallis test followed by Dunn’s test to correct multiple comparisons (two-sided). Source data are provided in the Source Data file.

Techniques Used: Concentration Assay, Chromatin Immunoprecipitation

Specificity of digital nanopillar SERS platform for FGF-2 cytokine detection. Representative confocal SERS images in the presence of a target FGF-2 (1031 aM), and negative controls with non-target controls b G-CSF (1031 aM), c GM-CSF (1031 aM), d CX3CL1 (1031 aM), and e PBS. The median (interquartile range) of active pillars per scanning image for FGF-2, G-CSF, GM-CSF, CX3CL1, and PBS was 72 (63.5–76.75), 1.5 (1.5–2), 2 (1–4), 0.5 (0–1.25), and 1 (1–1.75), respectively. Data from one independent experiment.
Figure Legend Snippet: Specificity of digital nanopillar SERS platform for FGF-2 cytokine detection. Representative confocal SERS images in the presence of a target FGF-2 (1031 aM), and negative controls with non-target controls b G-CSF (1031 aM), c GM-CSF (1031 aM), d CX3CL1 (1031 aM), and e PBS. The median (interquartile range) of active pillars per scanning image for FGF-2, G-CSF, GM-CSF, CX3CL1, and PBS was 72 (63.5–76.75), 1.5 (1.5–2), 2 (1–4), 0.5 (0–1.25), and 1 (1–1.75), respectively. Data from one independent experiment.

Techniques Used:

35) Product Images from "Glutathionylation chemistry promotes interleukin-1 beta-mediated glycolytic reprogramming and pro-inflammatory signaling in lung epithelial cells"

Article Title: Glutathionylation chemistry promotes interleukin-1 beta-mediated glycolytic reprogramming and pro-inflammatory signaling in lung epithelial cells

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

doi: 10.1096/fj.202002687RR

Glutaredoxin absence exacerbates pro-inflammatory signaling induced by IL1B in airway basal cells. A, Gene expression of Tslp , Csf2 , Ccl20 , and Cxcl1 in WT and Glrx −/− control airway basal cell or 24 hours after stimulation with IL1B. B, Secreted pro-inflammatory cytokines TSLP, GM-CSF, CCL20, and KC levels measured in culture media supernatant 24 hours poststimulation with IL1B. A 10 mM of 2-DG was used for 1 hour before IL1B stimulation
Figure Legend Snippet: Glutaredoxin absence exacerbates pro-inflammatory signaling induced by IL1B in airway basal cells. A, Gene expression of Tslp , Csf2 , Ccl20 , and Cxcl1 in WT and Glrx −/− control airway basal cell or 24 hours after stimulation with IL1B. B, Secreted pro-inflammatory cytokines TSLP, GM-CSF, CCL20, and KC levels measured in culture media supernatant 24 hours poststimulation with IL1B. A 10 mM of 2-DG was used for 1 hour before IL1B stimulation

Techniques Used: Expressing

36) Product Images from "Exosomal lncRNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1)contributes to the progression of allergic rhinitis via modulating microRNA-511/Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) axis"

Article Title: Exosomal lncRNA Nuclear Paraspeckle Assembly Transcript 1 (NEAT1)contributes to the progression of allergic rhinitis via modulating microRNA-511/Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) axis

Journal: Bioengineered

doi: 10.1080/21655979.2021.1982313

NEAT1 knockdown regulates IL-13-triggered inflammatory cytokine, mucus production, and apoptosis in HNECs. (a). RT-qPCR showed the level of NEAT1 in human HNECstransfected with si-NEAT1 or si-NC. (b–g) RT-qPCR and ELISA assay showed the expression levels of GM-CSF, eotaxin-1, and MUC5AC in IL-13-stimulated HNECs transfected with si-NEAT1 or si-NC. (h and i) CCK-8 and TUNEL assays indicated the cell viability and apoptosis in IL-13-treated HNECs transfected with si-NEAT1 or si-NC. *P
Figure Legend Snippet: NEAT1 knockdown regulates IL-13-triggered inflammatory cytokine, mucus production, and apoptosis in HNECs. (a). RT-qPCR showed the level of NEAT1 in human HNECstransfected with si-NEAT1 or si-NC. (b–g) RT-qPCR and ELISA assay showed the expression levels of GM-CSF, eotaxin-1, and MUC5AC in IL-13-stimulated HNECs transfected with si-NEAT1 or si-NC. (h and i) CCK-8 and TUNEL assays indicated the cell viability and apoptosis in IL-13-treated HNECs transfected with si-NEAT1 or si-NC. *P

Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, CCK-8 Assay, TUNEL Assay

miR-511 is a downstream regulator of NEAT1 in AR. (a) RT-qPCR showed miR-511 expression in HNECs transfected with si-NC, si-NEAT1, and si-NEAT1+miR-511 inhibitor. (b–g) RT-qPCR and ELISA assay showed the expression levels of GM-CSF, eotaxin-1, and MUC5AC in IL-13-treated HNECs transfected with si-NC, si-NEAT1, and si-NEAT1+ miR-511 inhibitor. (h and i) CCK-8 and TUNEL assays indicated the cell viability and apoptosis in IL-13-treated HNECs transfected with si-NC, si-NEAT1, and si-NEAT1+ miR-511 inhibitor. *P
Figure Legend Snippet: miR-511 is a downstream regulator of NEAT1 in AR. (a) RT-qPCR showed miR-511 expression in HNECs transfected with si-NC, si-NEAT1, and si-NEAT1+miR-511 inhibitor. (b–g) RT-qPCR and ELISA assay showed the expression levels of GM-CSF, eotaxin-1, and MUC5AC in IL-13-treated HNECs transfected with si-NC, si-NEAT1, and si-NEAT1+ miR-511 inhibitor. (h and i) CCK-8 and TUNEL assays indicated the cell viability and apoptosis in IL-13-treated HNECs transfected with si-NC, si-NEAT1, and si-NEAT1+ miR-511 inhibitor. *P

Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, TUNEL Assay

NEAT1 silence attenuates the exosome-induced inflammatory response and apoptosis of HNECs. (a) RT-qPCR showed NEAT1 level in HNECs treated with PBS, exosome, exosome+si-NC, and exosome+si-NEAT1. (b–g) RT-qPCR and ELISA assay showed the mRNA expression levels of GM-CSF, eotaxin-1, and MUC5AC in HNECs treated with exosome, exosome+si-NC, and exosome+si-NEAT1. (h and i) CCK-8 and TUNEL assays indicated cell viability and apoptosis in different groups. *P
Figure Legend Snippet: NEAT1 silence attenuates the exosome-induced inflammatory response and apoptosis of HNECs. (a) RT-qPCR showed NEAT1 level in HNECs treated with PBS, exosome, exosome+si-NC, and exosome+si-NEAT1. (b–g) RT-qPCR and ELISA assay showed the mRNA expression levels of GM-CSF, eotaxin-1, and MUC5AC in HNECs treated with exosome, exosome+si-NC, and exosome+si-NEAT1. (h and i) CCK-8 and TUNEL assays indicated cell viability and apoptosis in different groups. *P

Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, CCK-8 Assay, TUNEL Assay

NEAT1 regulates IL-13-induced dysfunction of HNECs via miR-511/NR4A2 axis. (a) NR4A2 expression was detected by RT-qPCR in HNECs transfected with NC inhibitor, miR-511 inhibitor, miR-511 inhibitor+si-NR4A2. (b–g) The levels of GM-CSF, eotaxin-1, and MUC5AC were determined by RT-qPCR and ELISA in IL-13-treated HNECs transfected with NC inhibitor, miR-511 inhibitor, miR-511 inhibitor + si-NR4A2. (h and i) TUNEL and CCK-8 assays indicated the cell apoptosis and viability in IL-13-treated HNECs transfected with NC inhibitor, miR-511 inhibitor, miR-511 inhibitor + si-NR4A2. (j) NR4A2 expression was measured by RT-qPCR in HNECs transfected with pcDNA3.1, pcDNA3.1-NEAT1, pcDNA3.1-NEAT1+ miR-511 mimics. (k) Correlation analysis between NEAT1 and NR4A2 in nasal mucosal tissues from AR patients. *P
Figure Legend Snippet: NEAT1 regulates IL-13-induced dysfunction of HNECs via miR-511/NR4A2 axis. (a) NR4A2 expression was detected by RT-qPCR in HNECs transfected with NC inhibitor, miR-511 inhibitor, miR-511 inhibitor+si-NR4A2. (b–g) The levels of GM-CSF, eotaxin-1, and MUC5AC were determined by RT-qPCR and ELISA in IL-13-treated HNECs transfected with NC inhibitor, miR-511 inhibitor, miR-511 inhibitor + si-NR4A2. (h and i) TUNEL and CCK-8 assays indicated the cell apoptosis and viability in IL-13-treated HNECs transfected with NC inhibitor, miR-511 inhibitor, miR-511 inhibitor + si-NR4A2. (j) NR4A2 expression was measured by RT-qPCR in HNECs transfected with pcDNA3.1, pcDNA3.1-NEAT1, pcDNA3.1-NEAT1+ miR-511 mimics. (k) Correlation analysis between NEAT1 and NR4A2 in nasal mucosal tissues from AR patients. *P

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, TUNEL Assay, CCK-8 Assay

37) Product Images from "Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension"

Article Title: Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20111741

GM-CSF production in PAECs. (A and B) PAECs were transfected with BMPR2 siRNA (“B”) or nontargeting siRNA as a control (Con): 48 h later, BMPR2 mRNA was measured by qRT-PCR (A) and BMPR2 protein by immunoblot (B). 24 h after transfection with B or Con siRNA, PAECs were pretreated for 30 min with 100 ng/ml BMP-2 or vehicle before adding 10 ng/ml TNF. (C and D) 6 h later, GM-CSF mRNA normalized to GAPDH was determined by qRT-PCR (C) and GM-CSF–secreted protein was measured by ELISA (D). (E–J) Similar analyses were performed for IL-6 mRNA (E) and secreted protein (F), MCP-1 mRNA (G) and secreted protein (H), and IL-8 mRNA (I) and secreted protein (J). Bars are mean ± SEM for n = 3 (A–D) or n = 4 (E–J) experiments; *, P
Figure Legend Snippet: GM-CSF production in PAECs. (A and B) PAECs were transfected with BMPR2 siRNA (“B”) or nontargeting siRNA as a control (Con): 48 h later, BMPR2 mRNA was measured by qRT-PCR (A) and BMPR2 protein by immunoblot (B). 24 h after transfection with B or Con siRNA, PAECs were pretreated for 30 min with 100 ng/ml BMP-2 or vehicle before adding 10 ng/ml TNF. (C and D) 6 h later, GM-CSF mRNA normalized to GAPDH was determined by qRT-PCR (C) and GM-CSF–secreted protein was measured by ELISA (D). (E–J) Similar analyses were performed for IL-6 mRNA (E) and secreted protein (F), MCP-1 mRNA (G) and secreted protein (H), and IL-8 mRNA (I) and secreted protein (J). Bars are mean ± SEM for n = 3 (A–D) or n = 4 (E–J) experiments; *, P

Techniques Used: Transfection, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

38) Product Images from "Triggering Dectin-1-Pathway Alone Is Not Sufficient to Induce Cytokine Production by Murine Macrophages"

Article Title: Triggering Dectin-1-Pathway Alone Is Not Sufficient to Induce Cytokine Production by Murine Macrophages

Journal: PLoS ONE

doi: 10.1371/journal.pone.0148464

Sc cell wall crude products- and zymosan-induced cytokine productions are mainly dectin-1-dependent. WT and Clec7a -/- BMDM were stimulated with Sc extracts enriched in BG for 8 h. After incubation, cell culture supernatants were harvested and stored at -20°C. Chemokines and cytokines (TNFα, IL-6, CCL2, IL-10 and IL-1β) were quantified using a customized multiplex assay and GM-CSF was measured with a cytokine detection kit. Data are expressed as the mean ± SD of three independent experiments performed in triplicate. Mean values not sharing the same letter are significantly different according to the Student’s t-test ( p
Figure Legend Snippet: Sc cell wall crude products- and zymosan-induced cytokine productions are mainly dectin-1-dependent. WT and Clec7a -/- BMDM were stimulated with Sc extracts enriched in BG for 8 h. After incubation, cell culture supernatants were harvested and stored at -20°C. Chemokines and cytokines (TNFα, IL-6, CCL2, IL-10 and IL-1β) were quantified using a customized multiplex assay and GM-CSF was measured with a cytokine detection kit. Data are expressed as the mean ± SD of three independent experiments performed in triplicate. Mean values not sharing the same letter are significantly different according to the Student’s t-test ( p

Techniques Used: Incubation, Cell Culture, Multiplex Assay

39) Product Images from "Selenium Prevents Inflammation in Human Placenta and Adipose Tissue In Vitro: Implications for Metabolic Diseases of Pregnancy Associated with Inflammation"

Article Title: Selenium Prevents Inflammation in Human Placenta and Adipose Tissue In Vitro: Implications for Metabolic Diseases of Pregnancy Associated with Inflammation

Journal: Nutrients

doi: 10.3390/nu14163286

Effect of selenium on pro-inflammatory cytokine expression in VAT and SAT from pregnant women. ( A – E ) VAT ( n = 6 patients) and ( F – J ) SAT ( n = 6 patients) were incubated with or without 10 μM sodium selenite (Sel) for 1 h and then stimulated with 10 µg/mL LPS or 10 ng/mL TNF-α for a further 20 h. ( A – D , F – I ) GM-CSF, IL1-A, IL1-B and IL6 mRNA expression was analysed by RT-qPCR and fold change was calculated relative to basal expression. ( E , J ) The concentration of IL6 in the conditioned media was assayed by ELISA. For all graphs, individual data points represent six independent experiments and are displayed as mean ± SEM. a p ≤ 0.05 vs. LPS, b p ≤ 0.05 vs. TNF; repeated measures one-way ANOVA.
Figure Legend Snippet: Effect of selenium on pro-inflammatory cytokine expression in VAT and SAT from pregnant women. ( A – E ) VAT ( n = 6 patients) and ( F – J ) SAT ( n = 6 patients) were incubated with or without 10 μM sodium selenite (Sel) for 1 h and then stimulated with 10 µg/mL LPS or 10 ng/mL TNF-α for a further 20 h. ( A – D , F – I ) GM-CSF, IL1-A, IL1-B and IL6 mRNA expression was analysed by RT-qPCR and fold change was calculated relative to basal expression. ( E , J ) The concentration of IL6 in the conditioned media was assayed by ELISA. For all graphs, individual data points represent six independent experiments and are displayed as mean ± SEM. a p ≤ 0.05 vs. LPS, b p ≤ 0.05 vs. TNF; repeated measures one-way ANOVA.

Techniques Used: Expressing, Incubation, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Effect of selenium on pro-inflammatory cytokine expression in human placenta. Human placenta was incubated with or without 10 μM sodium selenite (Sel) for 1 h and then stimulated with 10 µg/mL LPS or 10 ng/mL TNF-α for a further 20 h ( n = 6 patients). ( A , B , D , E ) GM-CSF, IL1-A, IL1-B and IL6 mRNA expression were analysed by RT-qPCR and fold change was calculated relative to basal expression. ( C , F ) The concentration of IL1-A and IL6 in the conditioned media was assayed by ELISA. For all graphs, individual data points represent six independent experiments and are displayed as mean ± SEM. a p ≤ 0.05 vs. LPS, b p ≤ 0.05 vs. TNF-α; repeated measures one-way ANOVA.
Figure Legend Snippet: Effect of selenium on pro-inflammatory cytokine expression in human placenta. Human placenta was incubated with or without 10 μM sodium selenite (Sel) for 1 h and then stimulated with 10 µg/mL LPS or 10 ng/mL TNF-α for a further 20 h ( n = 6 patients). ( A , B , D , E ) GM-CSF, IL1-A, IL1-B and IL6 mRNA expression were analysed by RT-qPCR and fold change was calculated relative to basal expression. ( C , F ) The concentration of IL1-A and IL6 in the conditioned media was assayed by ELISA. For all graphs, individual data points represent six independent experiments and are displayed as mean ± SEM. a p ≤ 0.05 vs. LPS, b p ≤ 0.05 vs. TNF-α; repeated measures one-way ANOVA.

Techniques Used: Expressing, Incubation, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

40) Product Images from "Histamine Promotes the Development of Monocyte-Derived Dendritic Cells and Reduces Tumor Growth by Targeting the Myeloid NADPH Oxidase"

Article Title: Histamine Promotes the Development of Monocyte-Derived Dendritic Cells and Reduces Tumor Growth by Targeting the Myeloid NADPH Oxidase

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1402991

Histamine promotes the development of DCs from monocyte precursors. Human monocytes were cultured for 3–6 d in the absence (Ctl) or presence of histamine (his) in IL-4– and GM-CSF–containing media (DC) or in the absence of growth
Figure Legend Snippet: Histamine promotes the development of DCs from monocyte precursors. Human monocytes were cultured for 3–6 d in the absence (Ctl) or presence of histamine (his) in IL-4– and GM-CSF–containing media (DC) or in the absence of growth

Techniques Used: Cell Culture, CTL Assay

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    R&D Systems gm csf
    Allo-MDM and ConA-MDM display differences in kinetics and production of cytokines. To compare cytokine production in Allo-MDM and ConA-MDM cultures, PBMC from six histoincompatible donors were stimulated by either allogeneically matching different pairs within the group or stimulating cells from individuals mitogenically. Following adherence of activated PBMC, nonadherent cells were removed from the cultures at 24 h poststimulation, and supernatants were collected from the cultures at the indicated intervals. Parallel cultures of Allo-MDM (red), ConA-MDM (blue), and unstimulated PBMC (green) were established as described in Materials and Methods. Cytokine-specific ELISA analysis was used to determine cytokine expression kinetics in cell-free culture supernatants collected at 6, 12, 24, 36, and 48 h and at 3, 5, and 8 days postisolation. Cytokines analyzed include IFN-γ (A), <t>TNF-α</t> (B), IL-2 (C), IL-3 (D), <t>GM-CSF</t> (E), G-CSF (F), M-CSF (G), IL-10 (H), IL-13 (I), IL-7 (J), TGF-β (K), IL-1β (L), and IL-6 (M).
    Gm Csf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant mouse gm csf
    Expression of IL-10 in <t>TGF-β</t> DC transduced with Ad-IL-10. B10 bone marrow-derived DC were propagated in the presence of <t>GM-CSF</t> and TGF-β 1 for 5 days, and then transduced with (a) Ad-LacZ or (b) Ad-IL-10 at 50 MOI as described in the Materials and Methods. An avidin–biotin complex–alkaline phosphatase immunocytochemical staining procedure was used to identify intracellular IL-10, as seen in (b); counterstained with fast red. Magnification: × 70 (a); × 140 (b).
    Recombinant Mouse Gm Csf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Allo-MDM and ConA-MDM display differences in kinetics and production of cytokines. To compare cytokine production in Allo-MDM and ConA-MDM cultures, PBMC from six histoincompatible donors were stimulated by either allogeneically matching different pairs within the group or stimulating cells from individuals mitogenically. Following adherence of activated PBMC, nonadherent cells were removed from the cultures at 24 h poststimulation, and supernatants were collected from the cultures at the indicated intervals. Parallel cultures of Allo-MDM (red), ConA-MDM (blue), and unstimulated PBMC (green) were established as described in Materials and Methods. Cytokine-specific ELISA analysis was used to determine cytokine expression kinetics in cell-free culture supernatants collected at 6, 12, 24, 36, and 48 h and at 3, 5, and 8 days postisolation. Cytokines analyzed include IFN-γ (A), TNF-α (B), IL-2 (C), IL-3 (D), GM-CSF (E), G-CSF (F), M-CSF (G), IL-10 (H), IL-13 (I), IL-7 (J), TGF-β (K), IL-1β (L), and IL-6 (M).

    Journal: Journal of Virology

    Article Title: Reactivation of Latent Human Cytomegalovirus in CD14+ Monocytes Is Differentiation Dependent

    doi: 10.1128/JVI.75.16.7543-7554.2001

    Figure Lengend Snippet: Allo-MDM and ConA-MDM display differences in kinetics and production of cytokines. To compare cytokine production in Allo-MDM and ConA-MDM cultures, PBMC from six histoincompatible donors were stimulated by either allogeneically matching different pairs within the group or stimulating cells from individuals mitogenically. Following adherence of activated PBMC, nonadherent cells were removed from the cultures at 24 h poststimulation, and supernatants were collected from the cultures at the indicated intervals. Parallel cultures of Allo-MDM (red), ConA-MDM (blue), and unstimulated PBMC (green) were established as described in Materials and Methods. Cytokine-specific ELISA analysis was used to determine cytokine expression kinetics in cell-free culture supernatants collected at 6, 12, 24, 36, and 48 h and at 3, 5, and 8 days postisolation. Cytokines analyzed include IFN-γ (A), TNF-α (B), IL-2 (C), IL-3 (D), GM-CSF (E), G-CSF (F), M-CSF (G), IL-10 (H), IL-13 (I), IL-7 (J), TGF-β (K), IL-1β (L), and IL-6 (M).

    Article Snippet: For neutralization experiments, polyclonal neutralizing goat antibodies against human TNF-α, IL-1α, IL-2, TGF-β, GM-CSF, and IFN-γ (all from R & D Systems) were used to block production of the respective lymphokine in Allo-MDM cultures.

    Techniques: Enzyme-linked Immunosorbent Assay, Expressing

    Reactivation of latent HCMV in Allo-MDM is dependent on production of IFN-γ. Allo-MDM were established by mixing PBMC from two healthy blood donors ( n = 3 with duplicates), and reactivation of HCMV was detected by expression of the HCMV proteins IE and gB. (A) Confocal microscopy analysis of a representative sample of an Allo-MDM culture which reactivated HCMV. The cells expressed both HCMV IE (red) and gB (green). Magnification, ×630. (B) Reactivation of HCMV was inhibited in Allo-MDM, which were established in the presence of neutralizing antibodies to IFN-γ. In contrast, reactivation of virus was not affected by the addition of neutralizing antibodies directed against IL-1, IL-2, TNF-α, TGF-β, or GM-CSF at the time of establishment of the Allo-MDM cultures. (B) Percent HCMV IE-expressing cells in the Allo-MDM cultures at 52 days poststimulation.

    Journal: Journal of Virology

    Article Title: Reactivation of Latent Human Cytomegalovirus in CD14+ Monocytes Is Differentiation Dependent

    doi: 10.1128/JVI.75.16.7543-7554.2001

    Figure Lengend Snippet: Reactivation of latent HCMV in Allo-MDM is dependent on production of IFN-γ. Allo-MDM were established by mixing PBMC from two healthy blood donors ( n = 3 with duplicates), and reactivation of HCMV was detected by expression of the HCMV proteins IE and gB. (A) Confocal microscopy analysis of a representative sample of an Allo-MDM culture which reactivated HCMV. The cells expressed both HCMV IE (red) and gB (green). Magnification, ×630. (B) Reactivation of HCMV was inhibited in Allo-MDM, which were established in the presence of neutralizing antibodies to IFN-γ. In contrast, reactivation of virus was not affected by the addition of neutralizing antibodies directed against IL-1, IL-2, TNF-α, TGF-β, or GM-CSF at the time of establishment of the Allo-MDM cultures. (B) Percent HCMV IE-expressing cells in the Allo-MDM cultures at 52 days poststimulation.

    Article Snippet: For neutralization experiments, polyclonal neutralizing goat antibodies against human TNF-α, IL-1α, IL-2, TGF-β, GM-CSF, and IFN-γ (all from R & D Systems) were used to block production of the respective lymphokine in Allo-MDM cultures.

    Techniques: Expressing, Confocal Microscopy

    Expression of IL-10 in TGF-β DC transduced with Ad-IL-10. B10 bone marrow-derived DC were propagated in the presence of GM-CSF and TGF-β 1 for 5 days, and then transduced with (a) Ad-LacZ or (b) Ad-IL-10 at 50 MOI as described in the Materials and Methods. An avidin–biotin complex–alkaline phosphatase immunocytochemical staining procedure was used to identify intracellular IL-10, as seen in (b); counterstained with fast red. Magnification: × 70 (a); × 140 (b).

    Journal: Immunology

    Article Title: Contrasting effects of myeloid dendritic cells transduced with an adenoviral vector encoding interleukin-10 on organ allograft and tumour rejection

    doi: 10.1046/j.1365-2567.2000.00096.x

    Figure Lengend Snippet: Expression of IL-10 in TGF-β DC transduced with Ad-IL-10. B10 bone marrow-derived DC were propagated in the presence of GM-CSF and TGF-β 1 for 5 days, and then transduced with (a) Ad-LacZ or (b) Ad-IL-10 at 50 MOI as described in the Materials and Methods. An avidin–biotin complex–alkaline phosphatase immunocytochemical staining procedure was used to identify intracellular IL-10, as seen in (b); counterstained with fast red. Magnification: × 70 (a); × 140 (b).

    Article Snippet: The culture and selection procedures used to generate and purify DC were similar to those reported initially by Inaba et al . and were modified as described., Briefly, freshly isolated BM cells obtained from femurs of B10 or B6 mice were cultured in 24-well plates (2 × 106 /well) in 2 ml of RPMI-1640 (Life Technologies, Gaithersburg, MD) supplemented with antibiotics and 10% v/v fetal calf serum (FCS) (Nalgene, Miami, FL) (referred to subsequently as complete medium), 4 ng/ml recombinant mouse GM-CSF, and 0·2 ng/ml recombinant human TGF-β1 (R and D Systems, Minneapolis, MN).

    Techniques: Expressing, Transduction, Derivative Assay, Avidin-Biotin Assay, Staining