mouse igg2a  (Thermo Fisher)


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  • 95
    Name:
    Mouse IgG2a Purified
    Description:
    • Isotype Mouse IgG2a• Class Class I ASR • Statement ASR Analyte Specific Reagent Analytical and performance characteristics are not established • Buffer PBS• Preservative 0 1 Sodium Azide
    Catalog Number:
    mg2a00
    Price:
    None
    Applications:
    Cell Analysis|Flow Cytometry Antibodies & Secondary Detection|Flow Cytometry
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher mouse igg2a
    Qa-2 expression in the skin grafts. The expression of Qa-2 in the skin grafts was examined by immunohistochemistry with a primary antibody specific for Qa-2. Mouse <t>IgG2a</t> was used for negative control staining. (A, B) Grafts from pretransplantation (Pre-tx)
    • Isotype Mouse IgG2a• Class Class I ASR • Statement ASR Analyte Specific Reagent Analytical and performance characteristics are not established • Buffer PBS• Preservative 0 1 Sodium Azide
    https://www.bioz.com/result/mouse igg2a/product/Thermo Fisher
    Average 95 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    mouse igg2a - by Bioz Stars, 2020-09
    95/100 stars

    Images

    1) Product Images from "Dynamic Expression of Qa-2 during Acute Graft Rejection"

    Article Title: Dynamic Expression of Qa-2 during Acute Graft Rejection

    Journal: Molecular Medicine

    doi: 10.2119/molmed.2010.00133

    Qa-2 expression in the skin grafts. The expression of Qa-2 in the skin grafts was examined by immunohistochemistry with a primary antibody specific for Qa-2. Mouse IgG2a was used for negative control staining. (A, B) Grafts from pretransplantation (Pre-tx)
    Figure Legend Snippet: Qa-2 expression in the skin grafts. The expression of Qa-2 in the skin grafts was examined by immunohistochemistry with a primary antibody specific for Qa-2. Mouse IgG2a was used for negative control staining. (A, B) Grafts from pretransplantation (Pre-tx)

    Techniques Used: Expressing, Immunohistochemistry, Negative Control, Staining

    2) Product Images from "Role of selectins on IgE-mediated skin reaction"

    Article Title: Role of selectins on IgE-mediated skin reaction

    Journal: British Journal of Pharmacology

    doi: 10.1038/sj.bjp.0703739

    Effects of mouse E-, P- and L-selectin-IgG on IgE-mediated skin reaction in mice. (A) Effects on the increase of ear swelling. (B) Effects on neutrophil infiltration. (C) Effects on eosinophil infiltration. Each column represents the mean of five or six animals. Vertical bars indicate s.e.mean. * P
    Figure Legend Snippet: Effects of mouse E-, P- and L-selectin-IgG on IgE-mediated skin reaction in mice. (A) Effects on the increase of ear swelling. (B) Effects on neutrophil infiltration. (C) Effects on eosinophil infiltration. Each column represents the mean of five or six animals. Vertical bars indicate s.e.mean. * P

    Techniques Used: Mouse Assay

    3) Product Images from "The Roles of Integrins in Function of Human Neutrophils after Their Migration through Endothelium into Interstitial Matrix"

    Article Title: The Roles of Integrins in Function of Human Neutrophils after Their Migration through Endothelium into Interstitial Matrix

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0118593

    Effects of inhibition of integrin function on entry of neutrophils into collagen gels and depth of penetration induced by fMLP. Collagen gels were formed and loaded with (A) or confocal microscopy (B). B. Representative images of neutrophil nuclei (i) or intracellular bioparticles at 60min (ii) analysed by confocal microscopy., before neutrophils were added and allowed to settle and migrate for 30 and 60 min. A,C,E: Proportion of added neutrophils entering the gel. B,D,F: Average depth penetrated by the neutrophils that had entered. In A, B, neutrophils were untreated, or treated with antibody against β2-integrin or a small molecule inhibitor (CT7010). In C, D, neutrophils were untreated, or treated with antibody against β1 or against β3-integrin, or with RGDS peptide. In E, F, neutrophils were untreated, or pre-treated with antibodies against β1-, β2- and β3-integrin, or with a combination of non-specific IgG with matching isotypes. In A and E, ANOVA showed a significant effect of treatment on entry into the gel (p
    Figure Legend Snippet: Effects of inhibition of integrin function on entry of neutrophils into collagen gels and depth of penetration induced by fMLP. Collagen gels were formed and loaded with (A) or confocal microscopy (B). B. Representative images of neutrophil nuclei (i) or intracellular bioparticles at 60min (ii) analysed by confocal microscopy., before neutrophils were added and allowed to settle and migrate for 30 and 60 min. A,C,E: Proportion of added neutrophils entering the gel. B,D,F: Average depth penetrated by the neutrophils that had entered. In A, B, neutrophils were untreated, or treated with antibody against β2-integrin or a small molecule inhibitor (CT7010). In C, D, neutrophils were untreated, or treated with antibody against β1 or against β3-integrin, or with RGDS peptide. In E, F, neutrophils were untreated, or pre-treated with antibodies against β1-, β2- and β3-integrin, or with a combination of non-specific IgG with matching isotypes. In A and E, ANOVA showed a significant effect of treatment on entry into the gel (p

    Techniques Used: Inhibition, Confocal Microscopy

    Effects of inhibition of integrin function on adhesion to TNF-treated EC and migration into subendothelial matrix. HUVEC were stimulated with 100U/ml TNF for 4 h and neutrophils were added and allowed to adhere for 10 min before non-adherent cells were washed off and data collected at 15 min, 1 h, 3 h and 24 h. A,C: Total adherent neutrophils after 15min (% of those added). B,D: Average depth penetrated by the neutrophils that had transmigrated. In A, B, neutrophils were untreated, or pre-treated with antibody against β1- or against β3-integrin. In C, D, neutrophils were untreated, or pre-treated with antibodies against β1-, β2- and β3-integrin, or with a combination of non-specific IgG with matching isotypes. In C, ANOVA showed a significant effect of treatment (p
    Figure Legend Snippet: Effects of inhibition of integrin function on adhesion to TNF-treated EC and migration into subendothelial matrix. HUVEC were stimulated with 100U/ml TNF for 4 h and neutrophils were added and allowed to adhere for 10 min before non-adherent cells were washed off and data collected at 15 min, 1 h, 3 h and 24 h. A,C: Total adherent neutrophils after 15min (% of those added). B,D: Average depth penetrated by the neutrophils that had transmigrated. In A, B, neutrophils were untreated, or pre-treated with antibody against β1- or against β3-integrin. In C, D, neutrophils were untreated, or pre-treated with antibodies against β1-, β2- and β3-integrin, or with a combination of non-specific IgG with matching isotypes. In C, ANOVA showed a significant effect of treatment (p

    Techniques Used: Inhibition, Migration

    4) Product Images from "Active immunity and T-cell populations in pigs intraperitoneally inoculated with baculovirus-expressed transmissible gastroenteritis virus structural proteins"

    Article Title: Active immunity and T-cell populations in pigs intraperitoneally inoculated with baculovirus-expressed transmissible gastroenteritis virus structural proteins

    Journal: Veterinary Immunology and Immunopathology

    doi: 10.1016/S0165-2427(99)00074-4

    TGEV-specific IgG and IgA antibody secreting cells (ASC) as detected at PCD 0 and 6–7 from 2 to 8 pigs per time point of groups 1–4. A, B, C – in vivo ASC numbers for ileum lamina propria, mesenteric lymph node and peripheral blood mononuclear cells (MNC); D, E – in vitro memory ASC numbers (after 4–5 days of in vitro MNC stimulation with TGEV inactivated antigens) for mesenteric lymph node and peripheral blood lymphoid tissues. Differences in group ASC numbers were measured at PCD 0 and 6–7: * above the bars indicate significantly elevated ( p
    Figure Legend Snippet: TGEV-specific IgG and IgA antibody secreting cells (ASC) as detected at PCD 0 and 6–7 from 2 to 8 pigs per time point of groups 1–4. A, B, C – in vivo ASC numbers for ileum lamina propria, mesenteric lymph node and peripheral blood mononuclear cells (MNC); D, E – in vitro memory ASC numbers (after 4–5 days of in vitro MNC stimulation with TGEV inactivated antigens) for mesenteric lymph node and peripheral blood lymphoid tissues. Differences in group ASC numbers were measured at PCD 0 and 6–7: * above the bars indicate significantly elevated ( p

    Techniques Used: In Vivo, In Vitro

    5) Product Images from "Distinct regulation of MHC molecule expression on astrocytes and microglia during viral encephalomyelitis"

    Article Title: Distinct regulation of MHC molecule expression on astrocytes and microglia during viral encephalomyelitis

    Journal: Glia

    doi: 10.1002/glia.20538

    Delayed MHC class I expression on astrocytes compared with microglia. CNS cells from naive (N) and infected mice analyzed for CD45 and MHC class I expression by flow cytometry at the indicated days p.i. ( A ) Representative density plot depicting CD45 lo microglia in the R1 region and CD45 − GFP + astrocytes in the R2 region from brains of naïve mice; numbers indicate percentages of cells in these gates. ( B ) Density plots showing class I expression on CD45 lo microglia relative to CD45 hi cells gated on total CD45 + cells. Histograms show kinetics of class I expression on microglia ( C ; R1 gate) and astrocytes ( D ; R2 gate) indicated by solid black lines. Mouse IgG2a isotype control staining is indicated by thin dashed lines. Numbers within histograms represent percentage of glia with upregulated class I expression relative to naïve mice indicated by the dotted vertical line; numbers in brackets depict median fluorescent intensity (MFI) in the population with increased class I expression. Data are representative of 3–4 experiments.
    Figure Legend Snippet: Delayed MHC class I expression on astrocytes compared with microglia. CNS cells from naive (N) and infected mice analyzed for CD45 and MHC class I expression by flow cytometry at the indicated days p.i. ( A ) Representative density plot depicting CD45 lo microglia in the R1 region and CD45 − GFP + astrocytes in the R2 region from brains of naïve mice; numbers indicate percentages of cells in these gates. ( B ) Density plots showing class I expression on CD45 lo microglia relative to CD45 hi cells gated on total CD45 + cells. Histograms show kinetics of class I expression on microglia ( C ; R1 gate) and astrocytes ( D ; R2 gate) indicated by solid black lines. Mouse IgG2a isotype control staining is indicated by thin dashed lines. Numbers within histograms represent percentage of glia with upregulated class I expression relative to naïve mice indicated by the dotted vertical line; numbers in brackets depict median fluorescent intensity (MFI) in the population with increased class I expression. Data are representative of 3–4 experiments.

    Techniques Used: Expressing, Infection, Mouse Assay, Flow Cytometry, Staining

    MHC class II expression on microglia and astrocytes. CNS cells from uninfected and infected mice were analyzed for MHC class II expression on microglia and astrocytes as depicted in Fig. 4 . Histograms show kinetics of class II expression on microglia ( A ; R1 gate) and astrocytes ( B ; R2 gate). Rat IgG2b was used as isotype control Ab for setting thresholds for positive staining. Numbers within histograms represent percentage of class II + glia; numbers in brackets depict MFI of the class II + population. Plots are representative of 3–4 experiments.
    Figure Legend Snippet: MHC class II expression on microglia and astrocytes. CNS cells from uninfected and infected mice were analyzed for MHC class II expression on microglia and astrocytes as depicted in Fig. 4 . Histograms show kinetics of class II expression on microglia ( A ; R1 gate) and astrocytes ( B ; R2 gate). Rat IgG2b was used as isotype control Ab for setting thresholds for positive staining. Numbers within histograms represent percentage of class II + glia; numbers in brackets depict MFI of the class II + population. Plots are representative of 3–4 experiments.

    Techniques Used: Expressing, Infection, Mouse Assay, Staining

    MHC class I and class II expression on IFN‐γ treated astrocytes. Mixed glial cultures from neonatal FVB/N mice were analyzed for CD45 and class I (top panel) and class II (bottom panel) expression after treatment with or without 1 ng/mL IFN‐γ for 48 h. Cells were gated on microglia (CD45 + ; left panel) or astrocytes (CD45 − ; right panel). Microglia comprised ∼30% of the culture. Microglia MHC expression on untreated cells is indicated by solid gray lines and on IFN‐γ treated cells by solid black lines. Mouse IgG2a and Rat IgG2b, respectively, were used as isotype control Ab (dashed lines); control staining overlaps in untreated and treated cells. Numbers within histograms represent percentage of MHC + glia to the right (IFN‐γ treated) or above (untreated) the respective population; numbers in brackets reveal MFI in the MHC + population.
    Figure Legend Snippet: MHC class I and class II expression on IFN‐γ treated astrocytes. Mixed glial cultures from neonatal FVB/N mice were analyzed for CD45 and class I (top panel) and class II (bottom panel) expression after treatment with or without 1 ng/mL IFN‐γ for 48 h. Cells were gated on microglia (CD45 + ; left panel) or astrocytes (CD45 − ; right panel). Microglia comprised ∼30% of the culture. Microglia MHC expression on untreated cells is indicated by solid gray lines and on IFN‐γ treated cells by solid black lines. Mouse IgG2a and Rat IgG2b, respectively, were used as isotype control Ab (dashed lines); control staining overlaps in untreated and treated cells. Numbers within histograms represent percentage of MHC + glia to the right (IFN‐γ treated) or above (untreated) the respective population; numbers in brackets reveal MFI in the MHC + population.

    Techniques Used: Expressing, Mouse Assay, Staining

    6) Product Images from "Mucosal vaccine adjuvant cyclic di-GMP differentiates lung moDCs into Bcl6+ and Bcl6− mature moDCs to induce lung memory CD4+ TH cells and lung TFH cells respectively"

    Article Title: Mucosal vaccine adjuvant cyclic di-GMP differentiates lung moDCs into Bcl6+ and Bcl6− mature moDCs to induce lung memory CD4+ TH cells and lung TFH cells respectively

    Journal: bioRxiv

    doi: 10.1101/2020.06.04.135244

    HDM induces CD4 + memory T H cells in mLNs but not in the lungs from Bcl6 fl/fl CD11c cre mice. A . A cartoon of chronic HDM treatment in mice. n=3 mice/group. B . Flow cytometry analysis ( left ) and frequency ( right ) of CD4 + CD69 + CD49a + T RM in HDM-induced Bcl6 fl/fl and Bcl6 fl/fl CD11c cre mice on day 35. (n=3mice/group) Data are representative of three independent experiments. C-D . Lung cells ( C ) or mediastinal lymph nodes (mLNs) ( D ) from HDM treated mice on day 35 from ( A ) were stimulated with 25μg/ml HDM for 4 days in culture. T H 2 and T H 17 cytokines were measured in the supernatant by ELISA. Data are representative of three independent experiments. E . Serum anti-HDM IgG1 were determined in the HDM mice on day 35 from ( A ) by ELISA. Data are representative of three independent experiments. Graphs represent means ± standard error. The significance is determined by one-way ANOVA Tukey’s multiple comparison test ( C, D ) or unpaired Student’s t test ( B ). *p
    Figure Legend Snippet: HDM induces CD4 + memory T H cells in mLNs but not in the lungs from Bcl6 fl/fl CD11c cre mice. A . A cartoon of chronic HDM treatment in mice. n=3 mice/group. B . Flow cytometry analysis ( left ) and frequency ( right ) of CD4 + CD69 + CD49a + T RM in HDM-induced Bcl6 fl/fl and Bcl6 fl/fl CD11c cre mice on day 35. (n=3mice/group) Data are representative of three independent experiments. C-D . Lung cells ( C ) or mediastinal lymph nodes (mLNs) ( D ) from HDM treated mice on day 35 from ( A ) were stimulated with 25μg/ml HDM for 4 days in culture. T H 2 and T H 17 cytokines were measured in the supernatant by ELISA. Data are representative of three independent experiments. E . Serum anti-HDM IgG1 were determined in the HDM mice on day 35 from ( A ) by ELISA. Data are representative of three independent experiments. Graphs represent means ± standard error. The significance is determined by one-way ANOVA Tukey’s multiple comparison test ( C, D ) or unpaired Student’s t test ( B ). *p

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

    moDCs promote CDG adjuvant-induced lung mucosal IgA and CD4 memory T H responses. A-B . RelA fl/fl and RelA fl/fl CD11c cre mice were immunized ( i.n .) with two doses (14 days apart) of PspA or PspA plus CDG (5μg). Anti-PspA IgG in serum ( A ) and IgA in BALF ( B ) were determined by ELISA 28 days post-immunization. (n=3mice/group) Data are representative of three independent experiments. C-D . Lung cells ( D ) or splenocytes ( C ) from immunized RelA fl/fl and RelA fl/fl CD11c cre mice ( A ) were recalled with 5μg/ml PspA for 4 days in culture. Cytokines were measured in the supernatant by ELISA. E . Frequency of activated (CD86 + ) pulmonary DC subsets in RelA fl/fl and RelA fl/fl CD11c cre mice administered ( i.n .) with PBS or 5µg CDG for 16 hours. (n=3mice/group). Data are representative of two independent experiments. F . Experimental design for adoptive transfer. RelA fl/fl CD11c cre mice (n=3mice/group) were immunized with CDG/OVA. WT bone marrow Ly6C hi monocytes (1.5 million total cells) were transferred ( i.n .) at 30mins, 2hrs and 4hrs post-immunization. Mice were harvested on day 14. G . Lung cells from ( F ) were recalled with 2μg/ml OVA for 4 days in culture. Cytokines were measured in the supernatant by ELISA. Data are representative of three independent experiments. H . Frequency of lung T FH from ( F ). Data are representative of three independent experiments. I . CD4 + CD69 + CD49a + T RM were determine by flow cytometry from ( F ). Data are representative of three independent experiments. J . BALF anti-OVA IgA on day 14 from ( F ) were determined by ELISA. Data are representative of three independent experiments. Graphs represent the mean with error bars indication S.E.M. P values determined by one-way ANOVA Tukey’s multiple comparison test ( C, D, H ) or unpaired student t -test ( E, G ). * P
    Figure Legend Snippet: moDCs promote CDG adjuvant-induced lung mucosal IgA and CD4 memory T H responses. A-B . RelA fl/fl and RelA fl/fl CD11c cre mice were immunized ( i.n .) with two doses (14 days apart) of PspA or PspA plus CDG (5μg). Anti-PspA IgG in serum ( A ) and IgA in BALF ( B ) were determined by ELISA 28 days post-immunization. (n=3mice/group) Data are representative of three independent experiments. C-D . Lung cells ( D ) or splenocytes ( C ) from immunized RelA fl/fl and RelA fl/fl CD11c cre mice ( A ) were recalled with 5μg/ml PspA for 4 days in culture. Cytokines were measured in the supernatant by ELISA. E . Frequency of activated (CD86 + ) pulmonary DC subsets in RelA fl/fl and RelA fl/fl CD11c cre mice administered ( i.n .) with PBS or 5µg CDG for 16 hours. (n=3mice/group). Data are representative of two independent experiments. F . Experimental design for adoptive transfer. RelA fl/fl CD11c cre mice (n=3mice/group) were immunized with CDG/OVA. WT bone marrow Ly6C hi monocytes (1.5 million total cells) were transferred ( i.n .) at 30mins, 2hrs and 4hrs post-immunization. Mice were harvested on day 14. G . Lung cells from ( F ) were recalled with 2μg/ml OVA for 4 days in culture. Cytokines were measured in the supernatant by ELISA. Data are representative of three independent experiments. H . Frequency of lung T FH from ( F ). Data are representative of three independent experiments. I . CD4 + CD69 + CD49a + T RM were determine by flow cytometry from ( F ). Data are representative of three independent experiments. J . BALF anti-OVA IgA on day 14 from ( F ) were determined by ELISA. Data are representative of three independent experiments. Graphs represent the mean with error bars indication S.E.M. P values determined by one-way ANOVA Tukey’s multiple comparison test ( C, D, H ) or unpaired student t -test ( E, G ). * P

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

    Targeting soluble and transmembrane TNF to moDCs in vivo generate functionally distinct lung Bcl6 + and Bcl6 - moDCs respectively. A . Sorted naïve lung TNFR2 + cDC2 were adoptively transferred ( i.n .) into IRF4 fl/fl CD11c cre mice and immunized with CDG/PspA. On Day 14, Bcl6 + moDCs were determined by flow cytometry. (n=3 mice/group) Data are representative of three independent experiments. B . WT mice were treated ( i.n .) with 200ng recombinant TNF and PspA or PspA alone. On day 14, Bcl6 + moDCs in the lung were determined by Flow cytometry. (n=3 mice/group) Data are representative of two independent experiments. C . Cartoon illustrating the sTNF-Fc (IgG2A) and tmTNF-Fc (IgG2A) fusion proteins. D-F . IRF4 fl/fl CD11c cre mice were immunized ( i.n .) with CDG/NP 6 CGG and 100ng tmTNF-Fc (IgG2A) or 100ng sTNF-Fc (IgG2A). Lungs were harvested on day 14. Flow cytometry analysis of CCL20 ( D ), Bcl6 ( E ), and CXCL13 ( F ) expression by lung moDCs. (n=3 mice/group) Data are representative of three independent experiments. G-H . Flow cytometry analysis of lung T FH ( G ) and lung antigen-specific IgA ( H ) in mice from ( D-F ). Lungs were harvested on Day 14. Data are representative of three independent experiments. I . Flow cytometry analysis of lung CD4 + T RM cells in mice from (D-F) on day 14. Data are representative of three independent experiments. J . Lung cells from mice in (D-F) on day 14 post-immunization were recalled with 5μg/ml NP 6 CGG in culture for 4 days. Cytokines were measured in the supernatant by ELISA. Data are representative of three independent experiments. K . Flow cytometry analysis of lung CD4 + T cells in mice from (D-F) on day 14. Data are representative of three independent experiments. Graphs represent means ± standard error. The significance is represented by one-way ANOVA Tukey’s multiple comparison test. * p
    Figure Legend Snippet: Targeting soluble and transmembrane TNF to moDCs in vivo generate functionally distinct lung Bcl6 + and Bcl6 - moDCs respectively. A . Sorted naïve lung TNFR2 + cDC2 were adoptively transferred ( i.n .) into IRF4 fl/fl CD11c cre mice and immunized with CDG/PspA. On Day 14, Bcl6 + moDCs were determined by flow cytometry. (n=3 mice/group) Data are representative of three independent experiments. B . WT mice were treated ( i.n .) with 200ng recombinant TNF and PspA or PspA alone. On day 14, Bcl6 + moDCs in the lung were determined by Flow cytometry. (n=3 mice/group) Data are representative of two independent experiments. C . Cartoon illustrating the sTNF-Fc (IgG2A) and tmTNF-Fc (IgG2A) fusion proteins. D-F . IRF4 fl/fl CD11c cre mice were immunized ( i.n .) with CDG/NP 6 CGG and 100ng tmTNF-Fc (IgG2A) or 100ng sTNF-Fc (IgG2A). Lungs were harvested on day 14. Flow cytometry analysis of CCL20 ( D ), Bcl6 ( E ), and CXCL13 ( F ) expression by lung moDCs. (n=3 mice/group) Data are representative of three independent experiments. G-H . Flow cytometry analysis of lung T FH ( G ) and lung antigen-specific IgA ( H ) in mice from ( D-F ). Lungs were harvested on Day 14. Data are representative of three independent experiments. I . Flow cytometry analysis of lung CD4 + T RM cells in mice from (D-F) on day 14. Data are representative of three independent experiments. J . Lung cells from mice in (D-F) on day 14 post-immunization were recalled with 5μg/ml NP 6 CGG in culture for 4 days. Cytokines were measured in the supernatant by ELISA. Data are representative of three independent experiments. K . Flow cytometry analysis of lung CD4 + T cells in mice from (D-F) on day 14. Data are representative of three independent experiments. Graphs represent means ± standard error. The significance is represented by one-way ANOVA Tukey’s multiple comparison test. * p

    Techniques Used: In Vivo, Mouse Assay, Flow Cytometry, Recombinant, Expressing, Enzyme-linked Immunosorbent Assay

    7) Product Images from "Expression of the High Affinity IgE Receptor by Neutrophils of Individuals with Allergic Asthma is Both Minimal and Insensitive to Regulation by Serum IgE"

    Article Title: Expression of the High Affinity IgE Receptor by Neutrophils of Individuals with Allergic Asthma is Both Minimal and Insensitive to Regulation by Serum IgE

    Journal: Clinical immunology (Orlando, Fla.)

    doi: 10.1016/j.clim.2009.03.513

    Neutrophil FcεRI does not support IgE-mediated superoxide release or IgE-induced increase in neutrophil survival. (A) Neutrophils were incubated in buffer alone (Spont), with 10 µg/ml of anti-IgE, or with IgG or IgA immobilized in tissue
    Figure Legend Snippet: Neutrophil FcεRI does not support IgE-mediated superoxide release or IgE-induced increase in neutrophil survival. (A) Neutrophils were incubated in buffer alone (Spont), with 10 µg/ml of anti-IgE, or with IgG or IgA immobilized in tissue

    Techniques Used: Incubation

    8) Product Images from "SMURF1 silencing diminishes a CD44-high cancer stem cell-like population in head and neck squamous cell carcinoma"

    Article Title: SMURF1 silencing diminishes a CD44-high cancer stem cell-like population in head and neck squamous cell carcinoma

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-13-260

    BMP signaling and SMURF1 are differentially regulated in CD44 high /ALDH high sorted CSC-like populations. Representative FACS analyses of sorted cells are shown in A and B. (A) ALDEFLUOR stained cells in the presence of ALDH activity inhibitor DEAB and double-stained with APC-conjugated IgG isotype control. (B) Gating of CD44 low /ALDH low and CD44 high /ALDH high populations. (C–D) Immunoblotting of SMURF1, pSMAD1/5/8, and SMAD1 (as a control for total SMAD protein) in the sorted TR146 (C) and SCC-58 (D) cells shown as representatives of at least three repeats from two independent cell sorting experiments. CD44 + cells in the CD44 low /ALDH low sorted population were magnetically captured and removed to generate CD44 depleted /ALDH low cells. β-actin was used as a total protein loading control for all samples.
    Figure Legend Snippet: BMP signaling and SMURF1 are differentially regulated in CD44 high /ALDH high sorted CSC-like populations. Representative FACS analyses of sorted cells are shown in A and B. (A) ALDEFLUOR stained cells in the presence of ALDH activity inhibitor DEAB and double-stained with APC-conjugated IgG isotype control. (B) Gating of CD44 low /ALDH low and CD44 high /ALDH high populations. (C–D) Immunoblotting of SMURF1, pSMAD1/5/8, and SMAD1 (as a control for total SMAD protein) in the sorted TR146 (C) and SCC-58 (D) cells shown as representatives of at least three repeats from two independent cell sorting experiments. CD44 + cells in the CD44 low /ALDH low sorted population were magnetically captured and removed to generate CD44 depleted /ALDH low cells. β-actin was used as a total protein loading control for all samples.

    Techniques Used: FACS, Staining, Activity Assay

    9) Product Images from "Antibodies enhance infection of LSECs in a model of ADE-induced severe dengue disease"

    Article Title: Antibodies enhance infection of LSECs in a model of ADE-induced severe dengue disease

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2010.01.004

    Administration of anti-DENV antibody (15 μg IgG2a against DENV prM/M) induces a DHF/DSS-like disease in AG129 mice infected with 5×10 8 GE S221
    Figure Legend Snippet: Administration of anti-DENV antibody (15 μg IgG2a against DENV prM/M) induces a DHF/DSS-like disease in AG129 mice infected with 5×10 8 GE S221

    Techniques Used: Mouse Assay, Infection

    10) Product Images from "Tropism of Varicella-Zoster Virus for Human Tonsillar CD4+ T Lymphocytes That Express Activation, Memory, and Skin Homing Markers"

    Article Title: Tropism of Varicella-Zoster Virus for Human Tonsillar CD4+ T Lymphocytes That Express Activation, Memory, and Skin Homing Markers

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.22.11425-11433.2002

    Phenotypic analysis of CD4 + T cells from tonsils and peripheral blood. T cells isolated from peripheral blood (PBL [upper panels]) and tonsils (lower panels) were stained with anti-CD4, -CD45RA, -CD69, or -CD71 MAbs for FACS analysis. The cells were gated on CD45RA + CD4 + naive (shaded area) and CD45RA − CD4 + memory (thick line) cells and analyzed for expression of CD69 (A and B) and CD71 (C and D) in these subpopulations. T cells from the same preparation stained with mouse IgG2a were treated as an isotype control (dashed line).
    Figure Legend Snippet: Phenotypic analysis of CD4 + T cells from tonsils and peripheral blood. T cells isolated from peripheral blood (PBL [upper panels]) and tonsils (lower panels) were stained with anti-CD4, -CD45RA, -CD69, or -CD71 MAbs for FACS analysis. The cells were gated on CD45RA + CD4 + naive (shaded area) and CD45RA − CD4 + memory (thick line) cells and analyzed for expression of CD69 (A and B) and CD71 (C and D) in these subpopulations. T cells from the same preparation stained with mouse IgG2a were treated as an isotype control (dashed line).

    Techniques Used: Isolation, Staining, FACS, Expressing

    Chemotaxis of VZV-infected T cells in response to SDF-1α. Tonsillar T cells infected with VZV were tested for their chemotaxic response to human SDF-1α (100 nM) with 24-well plate tissue culture inserts with 5-μm-pore-size filters. All samples were performed in duplicative wells. Cells that were incubated with chemotaxis medium without containing chemokine were treated as negative controls. After 2 h of incubation at 37°C, cells that were in the starting population and that had migrated were harvested and stained with anti-CD4, anti-CD45RA, and anti-VZV IgG immune serum. The cells were gated on uninfected (VZV − CD4 + ) and infected (VZV + CD4 + ) T cells. The percentages of total CD4 + T cells (black bars), CD45RA + naive (dotted bars), and CD45RA − memory (hatched bars) CD4 + T cells that had migrated to SDF-1α from starting cell populations (maximal migration) in uninfected and infected cells were thus calculated and subtracted from background migration (range, 2.8 and 5.2%). The results shown are the averages of duplicate samples from three independent experiments.
    Figure Legend Snippet: Chemotaxis of VZV-infected T cells in response to SDF-1α. Tonsillar T cells infected with VZV were tested for their chemotaxic response to human SDF-1α (100 nM) with 24-well plate tissue culture inserts with 5-μm-pore-size filters. All samples were performed in duplicative wells. Cells that were incubated with chemotaxis medium without containing chemokine were treated as negative controls. After 2 h of incubation at 37°C, cells that were in the starting population and that had migrated were harvested and stained with anti-CD4, anti-CD45RA, and anti-VZV IgG immune serum. The cells were gated on uninfected (VZV − CD4 + ) and infected (VZV + CD4 + ) T cells. The percentages of total CD4 + T cells (black bars), CD45RA + naive (dotted bars), and CD45RA − memory (hatched bars) CD4 + T cells that had migrated to SDF-1α from starting cell populations (maximal migration) in uninfected and infected cells were thus calculated and subtracted from background migration (range, 2.8 and 5.2%). The results shown are the averages of duplicate samples from three independent experiments.

    Techniques Used: Chemotaxis Assay, Infection, Incubation, Staining, Migration

    FACS analysis of VZV-infected CD3 + T cells. Column-purified T cells isolated from tonsils were cocultured with VZV-infected HEL monolayers. The T cells were analyzed at days 2 to 3 postinfection and stained with anti-CD3 MAb and VZV IgG-negative human serum (A) as background staining or with anti-VZV IgG polyclonal immune serum (B) to detect VZV glycoproteins, which provide the indication of infection. The FACS plots show infection of tonsil T cells with VZV, and the frequency of CD3 + T cells that are VZV positive is as indicated.
    Figure Legend Snippet: FACS analysis of VZV-infected CD3 + T cells. Column-purified T cells isolated from tonsils were cocultured with VZV-infected HEL monolayers. The T cells were analyzed at days 2 to 3 postinfection and stained with anti-CD3 MAb and VZV IgG-negative human serum (A) as background staining or with anti-VZV IgG polyclonal immune serum (B) to detect VZV glycoproteins, which provide the indication of infection. The FACS plots show infection of tonsil T cells with VZV, and the frequency of CD3 + T cells that are VZV positive is as indicated.

    Techniques Used: FACS, Infection, Purification, Isolation, Staining

    PMA stimulation of VZV transcription in virally infected T cells. VZV-infected T cells at 2 days postinfection were preincubated either with 0.1% DMSO alone (A and B) or with U0126 at 10 μM in 0.1%DMSO (C) at 37°C for 30 min followed by PMA stimulation at 100 nM at 37°C for 24 h. The T cells were washed and stained with antihuman CD3 MAb and anti-VZV IgG immune serum for FACS analysis. The FACS plots show that VZV replication indicated by expression of VZV glycoproteins was enhanced by PMA treatment (B), but was inhibited by U0126 (C) compared to the unstimulated control (A).
    Figure Legend Snippet: PMA stimulation of VZV transcription in virally infected T cells. VZV-infected T cells at 2 days postinfection were preincubated either with 0.1% DMSO alone (A and B) or with U0126 at 10 μM in 0.1%DMSO (C) at 37°C for 30 min followed by PMA stimulation at 100 nM at 37°C for 24 h. The T cells were washed and stained with antihuman CD3 MAb and anti-VZV IgG immune serum for FACS analysis. The FACS plots show that VZV replication indicated by expression of VZV glycoproteins was enhanced by PMA treatment (B), but was inhibited by U0126 (C) compared to the unstimulated control (A).

    Techniques Used: Infection, Staining, FACS, Expressing

    11) Product Images from "Modulation of NKp30- and NKp46-Mediated Natural Killer Cell Responses by Poxviral Hemagglutinin"

    Article Title: Modulation of NKp30- and NKp46-Mediated Natural Killer Cell Responses by Poxviral Hemagglutinin

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002195

    Infection with vaccinia (VV) and ectromelia virus (ECTV) induces ligand structures for NKp30 and NKp46. Human HeLa cervix carcinoma (A, B) and mouse TC-1 lung carcinoma cells (C) were infected with VV strain WR (A, C) and ECTV strain MP-Nü (B) for 20 h, or left uninfected. Top row , infected ( black lines ) and uninfected cells ( gray lines ) were labeled with the anti-hemagglutinin (HA) mAb VVI-4G9 to control for the efficiency of infection, or goat anti-mouse Ig-PE secondary antibodies only ( light gray, filled ) and analyzed by cytofluorometry. 2nd to 4th row , Infected and uninfected cells were stained as indicated with soluble chimeric receptors consisting of the ectodomains of NKp30, NKp46 or NKp44, and the Fc portion of human IgG1. Stainings with goat anti-mouse or anti-human IgG-PE secondary antibody controls are shown as filled histograms.
    Figure Legend Snippet: Infection with vaccinia (VV) and ectromelia virus (ECTV) induces ligand structures for NKp30 and NKp46. Human HeLa cervix carcinoma (A, B) and mouse TC-1 lung carcinoma cells (C) were infected with VV strain WR (A, C) and ECTV strain MP-Nü (B) for 20 h, or left uninfected. Top row , infected ( black lines ) and uninfected cells ( gray lines ) were labeled with the anti-hemagglutinin (HA) mAb VVI-4G9 to control for the efficiency of infection, or goat anti-mouse Ig-PE secondary antibodies only ( light gray, filled ) and analyzed by cytofluorometry. 2nd to 4th row , Infected and uninfected cells were stained as indicated with soluble chimeric receptors consisting of the ectodomains of NKp30, NKp46 or NKp44, and the Fc portion of human IgG1. Stainings with goat anti-mouse or anti-human IgG-PE secondary antibody controls are shown as filled histograms.

    Techniques Used: Infection, Labeling, Staining

    12) Product Images from "Clarithromycin Suppresses Human Respiratory Syncytial Virus Infection-Induced Streptococcus pneumoniae Adhesion and Cytokine Production in a Pulmonary Epithelial Cell Line"

    Article Title: Clarithromycin Suppresses Human Respiratory Syncytial Virus Infection-Induced Streptococcus pneumoniae Adhesion and Cytokine Production in a Pulmonary Epithelial Cell Line

    Journal: Mediators of Inflammation

    doi: 10.1155/2012/528568

    Effect of CAM and PDTC on RSV-induced PAF receptor expression in A549 cells. One hour before RSV infection, CAM or PDTC is added to A549 cell culture at the indicated concentration. The cells were infected with the RSV at MOI of 1. After 24 h of infection, the cells were collected and then stained with an anti-PAF receptor antibody and phycoerythrin-labeled anti-mouse IgG antibody (thick lines). The stained cells were analyzed by flow cytometry. Thin lines indicate the cells stained with an unrelated isotype control antibody instead of the anti-PAF receptor antibody.
    Figure Legend Snippet: Effect of CAM and PDTC on RSV-induced PAF receptor expression in A549 cells. One hour before RSV infection, CAM or PDTC is added to A549 cell culture at the indicated concentration. The cells were infected with the RSV at MOI of 1. After 24 h of infection, the cells were collected and then stained with an anti-PAF receptor antibody and phycoerythrin-labeled anti-mouse IgG antibody (thick lines). The stained cells were analyzed by flow cytometry. Thin lines indicate the cells stained with an unrelated isotype control antibody instead of the anti-PAF receptor antibody.

    Techniques Used: Chick Chorioallantoic Membrane Assay, Expressing, Infection, Cell Culture, Concentration Assay, Staining, Labeling, Flow Cytometry, Cytometry

    13) Product Images from "Protection From Influenza by Intramuscular Gene Vector Delivery of a Broadly Neutralizing Nanobody Does Not Depend on Antibody Dependent Cellular Cytotoxicity"

    Article Title: Protection From Influenza by Intramuscular Gene Vector Delivery of a Broadly Neutralizing Nanobody Does Not Depend on Antibody Dependent Cellular Cytotoxicity

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00627

    R1a-B6 reformatted for in vivo gene delivery. (A) Surface structure model of hemagglutinin (HA) trimer of A(H1N1)pdm09 (PDB structure 3AL4) showing the key epitope residues of R1a-B6, Gly20, Trp21, and Ile45 (shown in red) located in the HA stem region (cyan) ( 40 ). The receptor binding site (magenta), fusion peptide (orange), and head domain (green) are also illustrated. (B) Four constructs, (i) R1a-B6 mouse Fc IgG1, (ii) R1a-B6 mouse Fc IgG2a, (iii) monovalent R1a-B6, and (iv) negative control mouse Fc IgG2a fusion carrying a nanobody, cAb1 [adapted from Arbabi Ghahroudi et al. ( 54 )], specific for chicken egg white lysozyme were produced in vitro . These constructs were cloned into an AAV expression system for protein expression in vivo . (C) Expression and purification of nanobody-Fc fusions. Detection of proteins was carried out under reducing and non-reducing conditions in SDS-PAGE gels. Theoretical molecular weights (MW) for R1a-B6-mIgG1, R1a-B6-mIgG2a, and cAb1-mIgG2a are ∼37 kDa under denaturing (reducing) conditions, and ∼75 kDA under non-reducing conditions. (D) In vitro ADCC activation. Activation of luciferase reporter gene is shown in relative luminescence units (RLU) as a function of nanobody-Fc concentration. Each well was measured in triplicate. (E) Binding of R1a-B6 against a broad range of influenza A subtypes as tested by ELISA. Half maximal effective concentration (EC 50 ) was measured in duplicate. There was no binding on A/TX/12(H3N2) or B/Brisbane/08 (data not shown). All values above the dotted line indicate no binding activity.
    Figure Legend Snippet: R1a-B6 reformatted for in vivo gene delivery. (A) Surface structure model of hemagglutinin (HA) trimer of A(H1N1)pdm09 (PDB structure 3AL4) showing the key epitope residues of R1a-B6, Gly20, Trp21, and Ile45 (shown in red) located in the HA stem region (cyan) ( 40 ). The receptor binding site (magenta), fusion peptide (orange), and head domain (green) are also illustrated. (B) Four constructs, (i) R1a-B6 mouse Fc IgG1, (ii) R1a-B6 mouse Fc IgG2a, (iii) monovalent R1a-B6, and (iv) negative control mouse Fc IgG2a fusion carrying a nanobody, cAb1 [adapted from Arbabi Ghahroudi et al. ( 54 )], specific for chicken egg white lysozyme were produced in vitro . These constructs were cloned into an AAV expression system for protein expression in vivo . (C) Expression and purification of nanobody-Fc fusions. Detection of proteins was carried out under reducing and non-reducing conditions in SDS-PAGE gels. Theoretical molecular weights (MW) for R1a-B6-mIgG1, R1a-B6-mIgG2a, and cAb1-mIgG2a are ∼37 kDa under denaturing (reducing) conditions, and ∼75 kDA under non-reducing conditions. (D) In vitro ADCC activation. Activation of luciferase reporter gene is shown in relative luminescence units (RLU) as a function of nanobody-Fc concentration. Each well was measured in triplicate. (E) Binding of R1a-B6 against a broad range of influenza A subtypes as tested by ELISA. Half maximal effective concentration (EC 50 ) was measured in duplicate. There was no binding on A/TX/12(H3N2) or B/Brisbane/08 (data not shown). All values above the dotted line indicate no binding activity.

    Techniques Used: In Vivo, Binding Assay, Construct, Negative Control, Produced, In Vitro, Clone Assay, Expressing, Purification, SDS Page, Activation Assay, Luciferase, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

    14) Product Images from "Identifying a Role for Toll-Like Receptor 3 in the Innate Immune Response to Chlamydia muridarum Infection in Murine Oviduct Epithelial Cells"

    Article Title: Identifying a Role for Toll-Like Receptor 3 in the Innate Immune Response to Chlamydia muridarum Infection in Murine Oviduct Epithelial Cells

    Journal: Infection and Immunity

    doi: 10.1128/IAI.05549-11

    IFN-β enhances TLR2 protein function. (A) Histogram data of TLR3-deficient OE cells that were either untreated or treated with 50 U of recombinant IFN-β/ml prior to staining with PE-conjugated TLR2-specific or IgG2a isotype control monoclonal
    Figure Legend Snippet: IFN-β enhances TLR2 protein function. (A) Histogram data of TLR3-deficient OE cells that were either untreated or treated with 50 U of recombinant IFN-β/ml prior to staining with PE-conjugated TLR2-specific or IgG2a isotype control monoclonal

    Techniques Used: Recombinant, Staining

    15) Product Images from "Investigating the Impact of Delivery System Design on the Efficacy of Self-Amplifying RNA Vaccines"

    Article Title: Investigating the Impact of Delivery System Design on the Efficacy of Self-Amplifying RNA Vaccines

    Journal: Vaccines

    doi: 10.3390/vaccines8020212

    Immunogenicity of SAM-RVG vaccine delivered by different cationic carriers. Groups of ten BALB/c mice were immunized i.m. on days 0 and 28 with either 1.5 or 0.15 μg of self-amplifying RNA encoding the rabies virus G protein formulated in DOTAP polymeric nanoparticles (NPs), DOTAP Liposomes or DDA Liposomes, and compared to the commercial vaccine Rabipur (1/20 of human dose). GSK trademark CNE56 was used as a positive control. Specific IgG titers were measured by enzyme-linked immunosorbent assay (ELISA). For each group, there were 5 samples, each representing data from pools of two mice (depicted as circles), and the geometric mean titers (GMTs) are solid lines. Sera were collected and analyzed ( A ) 4 weeks after the first immunization and ( B ) 2 weeks after the second immunization. Titers
    Figure Legend Snippet: Immunogenicity of SAM-RVG vaccine delivered by different cationic carriers. Groups of ten BALB/c mice were immunized i.m. on days 0 and 28 with either 1.5 or 0.15 μg of self-amplifying RNA encoding the rabies virus G protein formulated in DOTAP polymeric nanoparticles (NPs), DOTAP Liposomes or DDA Liposomes, and compared to the commercial vaccine Rabipur (1/20 of human dose). GSK trademark CNE56 was used as a positive control. Specific IgG titers were measured by enzyme-linked immunosorbent assay (ELISA). For each group, there were 5 samples, each representing data from pools of two mice (depicted as circles), and the geometric mean titers (GMTs) are solid lines. Sera were collected and analyzed ( A ) 4 weeks after the first immunization and ( B ) 2 weeks after the second immunization. Titers

    Techniques Used: Mouse Assay, Positive Control, Enzyme-linked Immunosorbent Assay

    16) Product Images from "Escherichia coli Maltose-Binding Protein Induces M1 Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner"

    Article Title: Escherichia coli Maltose-Binding Protein Induces M1 Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms16059896

    Effects of MBP-induced activation and polarization of RAW264.7 cells via TLR2 and TLR4. RAW264.7 cells were pre-incubated with anti-TLR2 or anti-TLR4 (20 μg/mL) antibody for 2 h prior to the addition of MBP or LPS (5 μg/mL) for 48 h. Mouse IgG2a (20 µg/mL) was used as isotype control. ( A ) MBP-induced NO production in RAW264.7 cells was partially inhibited by either anti-TLR2 or anti-TLR4; ( B ) MBP-induced M1 polarization of RAW264.7 cells was greatly inhibited by either anti-TLR2 or anti-TLR4. Results were presented as mean ± SD from three independent experiments, each performed in triplicate. * p
    Figure Legend Snippet: Effects of MBP-induced activation and polarization of RAW264.7 cells via TLR2 and TLR4. RAW264.7 cells were pre-incubated with anti-TLR2 or anti-TLR4 (20 μg/mL) antibody for 2 h prior to the addition of MBP or LPS (5 μg/mL) for 48 h. Mouse IgG2a (20 µg/mL) was used as isotype control. ( A ) MBP-induced NO production in RAW264.7 cells was partially inhibited by either anti-TLR2 or anti-TLR4; ( B ) MBP-induced M1 polarization of RAW264.7 cells was greatly inhibited by either anti-TLR2 or anti-TLR4. Results were presented as mean ± SD from three independent experiments, each performed in triplicate. * p

    Techniques Used: Activation Assay, Incubation

    17) Product Images from "Functional dichotomy of Vδ2 γδ T cells in chronic hepatitis C virus infections: role in cytotoxicity but not for IFN-γ production"

    Article Title: Functional dichotomy of Vδ2 γδ T cells in chronic hepatitis C virus infections: role in cytotoxicity but not for IFN-γ production

    Journal: Scientific Reports

    doi: 10.1038/srep26296

    IFN-α could directly modulate Vδ2 T-cell phenotype and function. ( A ) PBMCs from HCs were stained with an unlabeled primary antibody against IFNAR2 or an isotype-matched control antibody (mouse IgG2a), and then by a phycoerythrin (PE)-conjugated anti- mouse IgG2a secondary antibody. IFNAR2 expression on Vδ2 T cells was analyzed. A representative staining profile is shown. ( B ) PBMCs of healthy blood donors were separated for Vδ2 T cells and non- Vδ2 T cells using magnetic beads. Purified cells were stained with anti- Vδ2 T mAb and anti-CD3 mAb and analyzed by flow cytometry. The representative dot plots from six experiments are shown. ( C–F ) Purified Vδ2 T cells were preincubated with or without IFN-α for 24 h. ( C,D ) Expression of activation markers CD38 ( C ), and cytolytic enzymes GrB ( D ) on Vδ2 T cells was assessed by flow cytometry. ( E,F ) Expression of CD107a ( E ) and IFN-γ ( F ) on Vδ2 T cells upon zoledronate stimulation was analyzed by flow cytometry. n = 6 for each group. ( G ) Purified Vδ2 T cells were cultured in medium containing PBS or 100 U ⁄ml IFN-α, a blocking antibody against type I IFN receptor (anti-IFNAR2, 5 μg ⁄ml) or an isotype control antibody was added to the medium. After 24 hr of culture, the expression of CD38 on Vδ2 T cells was assessed. n = 5. * p
    Figure Legend Snippet: IFN-α could directly modulate Vδ2 T-cell phenotype and function. ( A ) PBMCs from HCs were stained with an unlabeled primary antibody against IFNAR2 or an isotype-matched control antibody (mouse IgG2a), and then by a phycoerythrin (PE)-conjugated anti- mouse IgG2a secondary antibody. IFNAR2 expression on Vδ2 T cells was analyzed. A representative staining profile is shown. ( B ) PBMCs of healthy blood donors were separated for Vδ2 T cells and non- Vδ2 T cells using magnetic beads. Purified cells were stained with anti- Vδ2 T mAb and anti-CD3 mAb and analyzed by flow cytometry. The representative dot plots from six experiments are shown. ( C–F ) Purified Vδ2 T cells were preincubated with or without IFN-α for 24 h. ( C,D ) Expression of activation markers CD38 ( C ), and cytolytic enzymes GrB ( D ) on Vδ2 T cells was assessed by flow cytometry. ( E,F ) Expression of CD107a ( E ) and IFN-γ ( F ) on Vδ2 T cells upon zoledronate stimulation was analyzed by flow cytometry. n = 6 for each group. ( G ) Purified Vδ2 T cells were cultured in medium containing PBS or 100 U ⁄ml IFN-α, a blocking antibody against type I IFN receptor (anti-IFNAR2, 5 μg ⁄ml) or an isotype control antibody was added to the medium. After 24 hr of culture, the expression of CD38 on Vδ2 T cells was assessed. n = 5. * p

    Techniques Used: Staining, Expressing, Magnetic Beads, Purification, Flow Cytometry, Cytometry, Activation Assay, Cell Culture, Blocking Assay

    18) Product Images from "Natural Killer Cell Evasion Is Essential for Infection by Rhesus Cytomegalovirus"

    Article Title: Natural Killer Cell Evasion Is Essential for Infection by Rhesus Cytomegalovirus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005868

    MICB is retained in the ER and associates with Rh159 in RhCMV infected cells. A) Immature MICB is enriched in RhCMV-infected cells. U373-MICB cells were infected with RhCMV (MOI = 3) for 12 or 24 h or left uninfected prior to lysis in 1% NP40. Cell lysates were treated with Endoglycosidase H (E), PNGase F (P), or digestion buffer alone (-), separated by SDS-PAGE and immunoblotted with monoclonal antibodies (mAbs) to MICB or GAPDH. Mature glycosylated MICB (67kDa) is EndoH resistant (R), whereas immature MICB (I) remains EndoH sensitive with an apparent MW of 43kDa upon EndoH treatment (S) indicative of ER retention. B) RhCMV inhibits intracellular transport of MICB. U373-MICBs were infected with RhCMV (WT) (MOI = 3) for 24 h (visualization by light microscopy confirmed 100% cytopathic effect (CPE)) or left uninfected (NI) followed by metabolically labeling with [35S]cysteine and [35S]methionine for 30 min. The label was then chased for the indicated times (h), cells were lysed and MICB was immunoprecipitated from cell lysates using a MICB specific antibody. Precipitates were either digested with EndoH (+) or mock treated (-) followed by SDS-PAGE and autoradiography. Stars (*) indicate an EndoH-sensitive protein co-precipitating with MICB in infected cells. C-D) Isolation and identification of Rh159 co-immunoprecipitating with MICB. U373-MICB cells were infected with RhCMV (WT) or left non-infected (NI) as above and cells were lysed at 48 hpi. MICB was immunoprecipitated with anti–MICB mAb, the immunoprecipitates were separated by SDS-PAGE, and proteins visualized by Coomassie Blue staining. The IgG heavy chain (55kDa) is indicated. The 43kDa protein (*) was excised from the gel and digested with trypsin. D) Mass-spectrometric analysis by LC-MS/MS identified tryptic peptides corresponding to Rh159 (gray shaded boxes). The predicted amino acid sequence of Rh159 contains a signal sequence (italics), N-linked glycosylation sites (underlined), a C-terminal transmembrane anchor (bold), and an RXR ER retrieval motif (red). The results shown in A and B are representative of two or more independent experiments.
    Figure Legend Snippet: MICB is retained in the ER and associates with Rh159 in RhCMV infected cells. A) Immature MICB is enriched in RhCMV-infected cells. U373-MICB cells were infected with RhCMV (MOI = 3) for 12 or 24 h or left uninfected prior to lysis in 1% NP40. Cell lysates were treated with Endoglycosidase H (E), PNGase F (P), or digestion buffer alone (-), separated by SDS-PAGE and immunoblotted with monoclonal antibodies (mAbs) to MICB or GAPDH. Mature glycosylated MICB (67kDa) is EndoH resistant (R), whereas immature MICB (I) remains EndoH sensitive with an apparent MW of 43kDa upon EndoH treatment (S) indicative of ER retention. B) RhCMV inhibits intracellular transport of MICB. U373-MICBs were infected with RhCMV (WT) (MOI = 3) for 24 h (visualization by light microscopy confirmed 100% cytopathic effect (CPE)) or left uninfected (NI) followed by metabolically labeling with [35S]cysteine and [35S]methionine for 30 min. The label was then chased for the indicated times (h), cells were lysed and MICB was immunoprecipitated from cell lysates using a MICB specific antibody. Precipitates were either digested with EndoH (+) or mock treated (-) followed by SDS-PAGE and autoradiography. Stars (*) indicate an EndoH-sensitive protein co-precipitating with MICB in infected cells. C-D) Isolation and identification of Rh159 co-immunoprecipitating with MICB. U373-MICB cells were infected with RhCMV (WT) or left non-infected (NI) as above and cells were lysed at 48 hpi. MICB was immunoprecipitated with anti–MICB mAb, the immunoprecipitates were separated by SDS-PAGE, and proteins visualized by Coomassie Blue staining. The IgG heavy chain (55kDa) is indicated. The 43kDa protein (*) was excised from the gel and digested with trypsin. D) Mass-spectrometric analysis by LC-MS/MS identified tryptic peptides corresponding to Rh159 (gray shaded boxes). The predicted amino acid sequence of Rh159 contains a signal sequence (italics), N-linked glycosylation sites (underlined), a C-terminal transmembrane anchor (bold), and an RXR ER retrieval motif (red). The results shown in A and B are representative of two or more independent experiments.

    Techniques Used: Infection, Lysis, SDS Page, Light Microscopy, Metabolic Labelling, Labeling, Immunoprecipitation, Autoradiography, Isolation, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing

    Rh159 interferes with intracellular transport of NKG2DL. A) Association with Rh159 prevents intracellular transport of MICB. U373-MICB cells were transduced with adenovectors (MOI = 80) expressing either GFP (AdGFP) or FLAG-tagged Rh159 (AdRh159FL) under control of tetracycline-dependent transactivator provided by co-transduced AdtTA (MOI = 20). At 24 hpi cells were metabolically labeled for 30 min with [35S]cysteine + [35S]methionine. Upon chasing the label for the indicated times (h), cells were lysed and MICB was immunoprecipitated with anti–MICB mAb. Precipitates were either digested with EndoH (+) or mock treated (-) followed by SDS-PAGE and autoradiography. (S) indicates EndoH-deglycosylated proteins. B) Rh159 co-immunoprecipitates with MICB. U373-ULBP3 (ULBP3, left panel) or U373-MICB (MICB, right panel) cells were lysed at 48 h post-transduction with AdRh159FL (Rh159) or an adenovector expressing FLAG-tagged SVV ORF 61 (SVV61) used as a negative control. MICB and ULBP3 were immunoprecipitated with anti–MICB and anti-ULBP3 mouse and goat mAbs, respectively, then immunoblotted with mouse anti-FLAG mAb. The mouse IgG heavy chain (55kDa) is indicated (HC). Input lanes were loaded with 10% total lysate used in immunoprecipitation and immunoblotted with mAbs for FLAG and GAPDH. The results shown are representative of two independent experiments. C) Rh159 reduces steady state levels of MICB. U373-MICB cells were lysed at 48 h post-transduction with the indicated Ad-vectors. Lysates were digested with EndoH (+) or mock treated (-) then immunoblotted with mAbs for MICB, FLAG or GAPDH. Note that both MICB and Rh159 are EndoH sensitive consistent with ER localization. The results shown are representative of two independent experiments. D-E) Rh159 reduces surface expression of MICA, MICB, ULBP1 and ULBP2 but not ULBP3. U373-NKG2DL cells were transduced with AdRh159FL or AdGFP as in A) but for 48 h. Cells were then lysed and immunoblotted with mAbs for FLAG and GAPDH ( D ), or stained with antibodies specific for the indicated proteins, or isotype control (dotted) and analyzed by flow cytometry. The results shown are representative of three or more independent experiments.
    Figure Legend Snippet: Rh159 interferes with intracellular transport of NKG2DL. A) Association with Rh159 prevents intracellular transport of MICB. U373-MICB cells were transduced with adenovectors (MOI = 80) expressing either GFP (AdGFP) or FLAG-tagged Rh159 (AdRh159FL) under control of tetracycline-dependent transactivator provided by co-transduced AdtTA (MOI = 20). At 24 hpi cells were metabolically labeled for 30 min with [35S]cysteine + [35S]methionine. Upon chasing the label for the indicated times (h), cells were lysed and MICB was immunoprecipitated with anti–MICB mAb. Precipitates were either digested with EndoH (+) or mock treated (-) followed by SDS-PAGE and autoradiography. (S) indicates EndoH-deglycosylated proteins. B) Rh159 co-immunoprecipitates with MICB. U373-ULBP3 (ULBP3, left panel) or U373-MICB (MICB, right panel) cells were lysed at 48 h post-transduction with AdRh159FL (Rh159) or an adenovector expressing FLAG-tagged SVV ORF 61 (SVV61) used as a negative control. MICB and ULBP3 were immunoprecipitated with anti–MICB and anti-ULBP3 mouse and goat mAbs, respectively, then immunoblotted with mouse anti-FLAG mAb. The mouse IgG heavy chain (55kDa) is indicated (HC). Input lanes were loaded with 10% total lysate used in immunoprecipitation and immunoblotted with mAbs for FLAG and GAPDH. The results shown are representative of two independent experiments. C) Rh159 reduces steady state levels of MICB. U373-MICB cells were lysed at 48 h post-transduction with the indicated Ad-vectors. Lysates were digested with EndoH (+) or mock treated (-) then immunoblotted with mAbs for MICB, FLAG or GAPDH. Note that both MICB and Rh159 are EndoH sensitive consistent with ER localization. The results shown are representative of two independent experiments. D-E) Rh159 reduces surface expression of MICA, MICB, ULBP1 and ULBP2 but not ULBP3. U373-NKG2DL cells were transduced with AdRh159FL or AdGFP as in A) but for 48 h. Cells were then lysed and immunoblotted with mAbs for FLAG and GAPDH ( D ), or stained with antibodies specific for the indicated proteins, or isotype control (dotted) and analyzed by flow cytometry. The results shown are representative of three or more independent experiments.

    Techniques Used: Transduction, Expressing, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Autoradiography, Negative Control, Staining, Flow Cytometry, Cytometry

    19) Product Images from "Fosfomycin suppresses RS-virus-induced Streptococcus pneumoniae and Haemophilus influenzae adhesion to respiratory epithelial cells via the platelet-activating factor receptor"

    Article Title: Fosfomycin suppresses RS-virus-induced Streptococcus pneumoniae and Haemophilus influenzae adhesion to respiratory epithelial cells via the platelet-activating factor receptor

    Journal: FEMS Microbiology Letters

    doi: 10.1111/j.1574-6968.2010.02049.x

    Upregulation of the PAF receptor (PAF-r) by RSV infection in A549 cells, as determined by flow cytometry (a). Suppression of RSV-induced PAF-r expression by fosfomycin (b and c). (a) A549 cells were infected with the RSV strain Long at MOI 1. After infection at the time indicated, the cells were collected and then stained with an anti-PAF-r antibody and a phycoerythrin-labeled anti-mouse IgG antibody (thick lines). The stained cells were analyzed by flow cytometry. Thin lines indicate cells stained with an unrelated isotype control antibody instead of the anti-PAF-r antibody. (b) Dose dependence of fosfomycin. Two hours before RSV infection, fosfomycin or PDTC was added at the indicated concentration. After a 24-h infection, the expression levels of the PAF-r were determined by flow cytometry as in (a). PDTC, an NF-κB inhibitor, was used as a control. (c) Fosfomycin treatment schedule. Two hours before, simultaneously with, or 4 or 12 h after RSV infection, fosfomycin was added at a concentration of 100 μg mL −1 . After 24 h of infection, the expression level of the PAF-r was analyzed by flow cytometry as in (a).
    Figure Legend Snippet: Upregulation of the PAF receptor (PAF-r) by RSV infection in A549 cells, as determined by flow cytometry (a). Suppression of RSV-induced PAF-r expression by fosfomycin (b and c). (a) A549 cells were infected with the RSV strain Long at MOI 1. After infection at the time indicated, the cells were collected and then stained with an anti-PAF-r antibody and a phycoerythrin-labeled anti-mouse IgG antibody (thick lines). The stained cells were analyzed by flow cytometry. Thin lines indicate cells stained with an unrelated isotype control antibody instead of the anti-PAF-r antibody. (b) Dose dependence of fosfomycin. Two hours before RSV infection, fosfomycin or PDTC was added at the indicated concentration. After a 24-h infection, the expression levels of the PAF-r were determined by flow cytometry as in (a). PDTC, an NF-κB inhibitor, was used as a control. (c) Fosfomycin treatment schedule. Two hours before, simultaneously with, or 4 or 12 h after RSV infection, fosfomycin was added at a concentration of 100 μg mL −1 . After 24 h of infection, the expression level of the PAF-r was analyzed by flow cytometry as in (a).

    Techniques Used: Infection, Flow Cytometry, Expressing, Staining, Labeling, Concentration Assay

    Related Articles

    Negative Control:

    Article Title: Dynamic Expression of Qa-2 during Acute Graft Rejection
    Article Snippet: .. Mouse IgG2a (eBioscience) was used for negative control staining. .. Peripheral blood samples were taken with heparinized tubes when the mice were euthanized.

    Incubation:

    Article Title: Pro-inflammatory properties of stromal cell-derived factor-1 (CXCL12) in collagen-induced arthritis
    Article Snippet: .. Plates were then incubated for 2 h with biotinylated rat antibody to mouse total IgG, IgG2a, IgG2b or IgG1 (Zymed Laboratories, San Francisco, CA, USA). .. Plates were washed and incubated for 1 h with streptavidin-peroxidase.

    Purification:

    Article Title: Critical Role for Galectin-3 in Airway Inflammation and Bronchial Hyperresponsiveness in a Murine Model of Asthma
    Article Snippet: .. The concentration of each Ig subclass in the samples was determined with the computer program SoftMaxPro provided with the plate reader (Molecular Devices, Sunnyvale, CA) and was read off a standard curve generated by incubating several concentrations of purified mouse IgG1 or IgG2a in wells coated either with rat anti-mouse IgG1 or rat anti-mouse IgG2a , respectively (CalTag Laboratories, Burlingame, CA) followed by biotinylated antibodies as above. .. After a surgical plane of anesthesia was achieved, the trachea was cannulated with a 19-gauge tubing adaptor attached to polyethylene tubing that passed through the plethysmograph chamber and was attached to a four-way connector, which was connected to a rodent ventilator (model 683; Harvard Apparatus, South Natick, MA) and pressure transducer.

    Concentration Assay:

    Article Title: Critical Role for Galectin-3 in Airway Inflammation and Bronchial Hyperresponsiveness in a Murine Model of Asthma
    Article Snippet: .. The concentration of each Ig subclass in the samples was determined with the computer program SoftMaxPro provided with the plate reader (Molecular Devices, Sunnyvale, CA) and was read off a standard curve generated by incubating several concentrations of purified mouse IgG1 or IgG2a in wells coated either with rat anti-mouse IgG1 or rat anti-mouse IgG2a , respectively (CalTag Laboratories, Burlingame, CA) followed by biotinylated antibodies as above. .. After a surgical plane of anesthesia was achieved, the trachea was cannulated with a 19-gauge tubing adaptor attached to polyethylene tubing that passed through the plethysmograph chamber and was attached to a four-way connector, which was connected to a rodent ventilator (model 683; Harvard Apparatus, South Natick, MA) and pressure transducer.

    Generated:

    Article Title: Critical Role for Galectin-3 in Airway Inflammation and Bronchial Hyperresponsiveness in a Murine Model of Asthma
    Article Snippet: .. The concentration of each Ig subclass in the samples was determined with the computer program SoftMaxPro provided with the plate reader (Molecular Devices, Sunnyvale, CA) and was read off a standard curve generated by incubating several concentrations of purified mouse IgG1 or IgG2a in wells coated either with rat anti-mouse IgG1 or rat anti-mouse IgG2a , respectively (CalTag Laboratories, Burlingame, CA) followed by biotinylated antibodies as above. .. After a surgical plane of anesthesia was achieved, the trachea was cannulated with a 19-gauge tubing adaptor attached to polyethylene tubing that passed through the plethysmograph chamber and was attached to a four-way connector, which was connected to a rodent ventilator (model 683; Harvard Apparatus, South Natick, MA) and pressure transducer.

    other:

    Article Title: Adjuvant-dependent modulation of Th1 and Th2 responses to immunization with ?-amyloid
    Article Snippet: To detect mouse IgM, IgG1, IgG2a and IgG2b isotypes, anti-mouse Ig subclass-specific HRP-conjugated secondary antibodies (Zymed, San Francisco, CA) were used.

    Staining:

    Article Title: Dynamic Expression of Qa-2 during Acute Graft Rejection
    Article Snippet: .. Mouse IgG2a (eBioscience) was used for negative control staining. .. Peripheral blood samples were taken with heparinized tubes when the mice were euthanized.

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  • 92
    Thermo Fisher flow cytometry polyclonal igg anti np
    <t>IgG</t> anti-NP suppresses IgM anti-NP responses at all NP densities. C57BL/6 mice were immunized i.v. with 5 × 10 7 SRBC-NP with high (SRBC-NP high ), intermediate (SRBC-NP int ) or low (SRBC-NP low ) epitope density with or without 30 μg <t>polyclonal</t> IgG anti-NP. Mice immunized with 5 × 10 7 unconjugated SRBC or with 30 μg IgG-anti NP only were used as controls. NP-specific immune responses were analyzed in spleen and serum samples obtained 5 days after immunization. ( A ) Cells were initially gated for B220 + cells. Representative flow <t>cytometry</t> gating for B220 + GC (defined as GL7 high CD95 high ) and non-GC λ1 + NP + cells from mice immunized with SRBC-NP high (left), IgG anti-NP + SRBC-NP high (middle) and IgG anti-NP alone (right). ( B ) Frequency of GC and non-GC NP + λ1 + cells in total B220 + cells. ( C ) NP-specific IgM-secreting cells per spleen. ( D ) Serum IgM anti-NP levels (serum dilution in ELISA = 1:625). The dashed line indicates the mean value of mice immunized with unconjugated SRBC. Representative of four independent experiments with 4–5 mice per group. ns = p > 0.05, * p
    Flow Cytometry Polyclonal Igg Anti Np, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg1
    Chop negatively regulates T-bet expression. a Time-dependent expression (upper panel) and corresponding densitometry quantitation (lower panel) of T-bet in primed wild-type and Ddit3 −/− CD8 + T cells. Left: protein level (0–72 h); right: Tbx21 mRNA levels 48 h post-activation. CD8 + T cells were stimulated with plate-bound anti-CD3/CD28 ( n = 3). b Tbx21 and Ifng mRNA expression in activated CD8 + T cells infected with control retrovirus (Retro-Ctrl) or Ddit3 -expressing retrovirus (Retro-Chop). Cells were primed for 48 h and then infected for additional 48 h in the presence of the stimulating anti-CD3/CD28 antibodies ( n = 4). c Ifng , Il12b2 , Cbfa3 , and Cxcr3 mRNA levels in control vs. Ddit3 −/− CD8 + T cells primed as in a ( n = 5). d Predicted Chop-binding site in the Tbx21 promoter region (GGGTGCAATCTTC). e Chromatin immunoprecipitation assay for the endogenous binding of Chop to Tbx21 promoter in primed wild-type or Ddit3 −/− CD8 + T cells. Chop-binding activity was measured by real-time quantitative PCR, compared with <t>IgG</t> binding activity after normalizing to the activity of anti-H3 ( n = 4). f A dual luciferase system composed of 2x-CRE containing Firefly luciferase reporter and the control Renilla luciferase reporter was transfected into 293T cells in combination with Ddit3 -expressing or control vectors. n = 4 experimental repeats. g Expression of Chop (left) and T-bet (right) by fluorescence-activated cell sorter (FACS) upon transduction of primed CD8 + T cells with green fluorescent protein (GFP)-coding retroviruses containing control or 8x-CRE sequences. Cells were primed for 48 h and then infected for another 48 h in the presence of the stimulating anti-CD3/CD28 antibodies plus interleukin (IL)-2 (50 U/ml). n = 3 independent repeats. h Interferon-γ (IFNγ) levels in primed CD8 + T cells transduced with: (1) GFP/CD90.1-expressing control virus (Ctrl); (2) Chop/CD90.1-expressing virus and GFP-expressing control virus (Chop); (3) CD90.1-expressing control virus and T-bet/GFP-expressing virus (T-bet); or (4) Chop/CD90.1-expressing virus and T-bet/GFP-expressing virus (Chop/T-bet). Cells were primed for 24 h and then transduced for additional 72 h in the presence of stimulating antibodies plus IL-2 (50 U/ml). Then IFNγ levels were detected by FACS in gated CD90.1 + GFP + cells. Right: Representative FACS result; left: Merged results from three independent experiments. In the bar graphs showing mean ± s.e.m., * p
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    Thermo Fisher igg
    <t>CRPS</t> patient serum and IgM had pronociceptive effects in B cell deficient fracture mice. At 3 weeks after tibia fracture and casting (FX) muMT mice lacking B cells and IgM exhibited unilateral hindpaw von Frey allodynia ( A ) and unweighting ( B ). After intraperitoneal injection of early (1–12 months post injury) CRPS patient serum (0.5ml, I.P) or IgM (500ug/1ml, I.P.) into 3 weeks post FX muMT mice, the mice gradually developed increased allodynia and unweighting over the ensuing week, and consistent with the 6 day half-life of IgM, these pronociceptive effects resolved by 2 weeks post-injection. The pronociceptive effects of the CRPS serum were restricted to the FX limb and there was no serum effect on post FX hindpaw edema or warmth (data not shown). No pronociceptive effects were observed after intraperitoneal injection of early CRPS patient <t>IgG</t> (5mg/1ml, I.P.) or after injection of control subject serum (0.5ml, I.P.) in 3 weeks post-FX mice. Measurements for ( A ) represent the difference between the FX side and contralateral paw, thus a negative value represents a decrease in mechanical withdrawal thresholds on the affected side. Measurements for ( B ) represent weight-bearing on the FX hind limb as a ratio to half of the total bilateral hind limb loading, thus, a percentage lower than 100% represents hindpaw unweighting. A 2-way repeated measures analysis of variance was used to test the effects of each treatment group on the dependent variables over time, using a Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SEM, n = 5 patients per cohort and each patients serum or immunoglobulin was injected into 3 mice for a total n of 15 mice. #P
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    IgG anti-NP suppresses IgM anti-NP responses at all NP densities. C57BL/6 mice were immunized i.v. with 5 × 10 7 SRBC-NP with high (SRBC-NP high ), intermediate (SRBC-NP int ) or low (SRBC-NP low ) epitope density with or without 30 μg polyclonal IgG anti-NP. Mice immunized with 5 × 10 7 unconjugated SRBC or with 30 μg IgG-anti NP only were used as controls. NP-specific immune responses were analyzed in spleen and serum samples obtained 5 days after immunization. ( A ) Cells were initially gated for B220 + cells. Representative flow cytometry gating for B220 + GC (defined as GL7 high CD95 high ) and non-GC λ1 + NP + cells from mice immunized with SRBC-NP high (left), IgG anti-NP + SRBC-NP high (middle) and IgG anti-NP alone (right). ( B ) Frequency of GC and non-GC NP + λ1 + cells in total B220 + cells. ( C ) NP-specific IgM-secreting cells per spleen. ( D ) Serum IgM anti-NP levels (serum dilution in ELISA = 1:625). The dashed line indicates the mean value of mice immunized with unconjugated SRBC. Representative of four independent experiments with 4–5 mice per group. ns = p > 0.05, * p

    Journal: Scientific Reports

    Article Title: IgG-mediated immune suppression in mice is epitope specific except during high epitope density conditions

    doi: 10.1038/s41598-018-33087-6

    Figure Lengend Snippet: IgG anti-NP suppresses IgM anti-NP responses at all NP densities. C57BL/6 mice were immunized i.v. with 5 × 10 7 SRBC-NP with high (SRBC-NP high ), intermediate (SRBC-NP int ) or low (SRBC-NP low ) epitope density with or without 30 μg polyclonal IgG anti-NP. Mice immunized with 5 × 10 7 unconjugated SRBC or with 30 μg IgG-anti NP only were used as controls. NP-specific immune responses were analyzed in spleen and serum samples obtained 5 days after immunization. ( A ) Cells were initially gated for B220 + cells. Representative flow cytometry gating for B220 + GC (defined as GL7 high CD95 high ) and non-GC λ1 + NP + cells from mice immunized with SRBC-NP high (left), IgG anti-NP + SRBC-NP high (middle) and IgG anti-NP alone (right). ( B ) Frequency of GC and non-GC NP + λ1 + cells in total B220 + cells. ( C ) NP-specific IgM-secreting cells per spleen. ( D ) Serum IgM anti-NP levels (serum dilution in ELISA = 1:625). The dashed line indicates the mean value of mice immunized with unconjugated SRBC. Representative of four independent experiments with 4–5 mice per group. ns = p > 0.05, * p

    Article Snippet: Flow cytometry Polyclonal IgG anti-NP (prepared as described above) was biotinylated using EZ-Link Sulfo-NHS-LC- LC-Biotin (sulfosuccinimidyl-6-biotinamido-6-hexanamido hexaonate) (Thermo Scientific) according to the manufacturer’s instructions.

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

    Chop negatively regulates T-bet expression. a Time-dependent expression (upper panel) and corresponding densitometry quantitation (lower panel) of T-bet in primed wild-type and Ddit3 −/− CD8 + T cells. Left: protein level (0–72 h); right: Tbx21 mRNA levels 48 h post-activation. CD8 + T cells were stimulated with plate-bound anti-CD3/CD28 ( n = 3). b Tbx21 and Ifng mRNA expression in activated CD8 + T cells infected with control retrovirus (Retro-Ctrl) or Ddit3 -expressing retrovirus (Retro-Chop). Cells were primed for 48 h and then infected for additional 48 h in the presence of the stimulating anti-CD3/CD28 antibodies ( n = 4). c Ifng , Il12b2 , Cbfa3 , and Cxcr3 mRNA levels in control vs. Ddit3 −/− CD8 + T cells primed as in a ( n = 5). d Predicted Chop-binding site in the Tbx21 promoter region (GGGTGCAATCTTC). e Chromatin immunoprecipitation assay for the endogenous binding of Chop to Tbx21 promoter in primed wild-type or Ddit3 −/− CD8 + T cells. Chop-binding activity was measured by real-time quantitative PCR, compared with IgG binding activity after normalizing to the activity of anti-H3 ( n = 4). f A dual luciferase system composed of 2x-CRE containing Firefly luciferase reporter and the control Renilla luciferase reporter was transfected into 293T cells in combination with Ddit3 -expressing or control vectors. n = 4 experimental repeats. g Expression of Chop (left) and T-bet (right) by fluorescence-activated cell sorter (FACS) upon transduction of primed CD8 + T cells with green fluorescent protein (GFP)-coding retroviruses containing control or 8x-CRE sequences. Cells were primed for 48 h and then infected for another 48 h in the presence of the stimulating anti-CD3/CD28 antibodies plus interleukin (IL)-2 (50 U/ml). n = 3 independent repeats. h Interferon-γ (IFNγ) levels in primed CD8 + T cells transduced with: (1) GFP/CD90.1-expressing control virus (Ctrl); (2) Chop/CD90.1-expressing virus and GFP-expressing control virus (Chop); (3) CD90.1-expressing control virus and T-bet/GFP-expressing virus (T-bet); or (4) Chop/CD90.1-expressing virus and T-bet/GFP-expressing virus (Chop/T-bet). Cells were primed for 24 h and then transduced for additional 72 h in the presence of stimulating antibodies plus IL-2 (50 U/ml). Then IFNγ levels were detected by FACS in gated CD90.1 + GFP + cells. Right: Representative FACS result; left: Merged results from three independent experiments. In the bar graphs showing mean ± s.e.m., * p

    Journal: Nature Communications

    Article Title: ER stress-induced mediator C/EBP homologous protein thwarts effector T cell activity in tumors through T-bet repression

    doi: 10.1038/s41467-019-09263-1

    Figure Lengend Snippet: Chop negatively regulates T-bet expression. a Time-dependent expression (upper panel) and corresponding densitometry quantitation (lower panel) of T-bet in primed wild-type and Ddit3 −/− CD8 + T cells. Left: protein level (0–72 h); right: Tbx21 mRNA levels 48 h post-activation. CD8 + T cells were stimulated with plate-bound anti-CD3/CD28 ( n = 3). b Tbx21 and Ifng mRNA expression in activated CD8 + T cells infected with control retrovirus (Retro-Ctrl) or Ddit3 -expressing retrovirus (Retro-Chop). Cells were primed for 48 h and then infected for additional 48 h in the presence of the stimulating anti-CD3/CD28 antibodies ( n = 4). c Ifng , Il12b2 , Cbfa3 , and Cxcr3 mRNA levels in control vs. Ddit3 −/− CD8 + T cells primed as in a ( n = 5). d Predicted Chop-binding site in the Tbx21 promoter region (GGGTGCAATCTTC). e Chromatin immunoprecipitation assay for the endogenous binding of Chop to Tbx21 promoter in primed wild-type or Ddit3 −/− CD8 + T cells. Chop-binding activity was measured by real-time quantitative PCR, compared with IgG binding activity after normalizing to the activity of anti-H3 ( n = 4). f A dual luciferase system composed of 2x-CRE containing Firefly luciferase reporter and the control Renilla luciferase reporter was transfected into 293T cells in combination with Ddit3 -expressing or control vectors. n = 4 experimental repeats. g Expression of Chop (left) and T-bet (right) by fluorescence-activated cell sorter (FACS) upon transduction of primed CD8 + T cells with green fluorescent protein (GFP)-coding retroviruses containing control or 8x-CRE sequences. Cells were primed for 48 h and then infected for another 48 h in the presence of the stimulating anti-CD3/CD28 antibodies plus interleukin (IL)-2 (50 U/ml). n = 3 independent repeats. h Interferon-γ (IFNγ) levels in primed CD8 + T cells transduced with: (1) GFP/CD90.1-expressing control virus (Ctrl); (2) Chop/CD90.1-expressing virus and GFP-expressing control virus (Chop); (3) CD90.1-expressing control virus and T-bet/GFP-expressing virus (T-bet); or (4) Chop/CD90.1-expressing virus and T-bet/GFP-expressing virus (Chop/T-bet). Cells were primed for 24 h and then transduced for additional 72 h in the presence of stimulating antibodies plus IL-2 (50 U/ml). Then IFNγ levels were detected by FACS in gated CD90.1 + GFP + cells. Right: Representative FACS result; left: Merged results from three independent experiments. In the bar graphs showing mean ± s.e.m., * p

    Article Snippet: After de-paraffinization and antigen retrieval, sections were blocked in 5% goat serum and incubated overnight with mouse monoclonal anti-CD8 (1:100, IgG1, C8/144B, #108M-98, Cell Marque) and mouse monoclonal anti-Chop (1:100, IgG2b, 9C8, #ab11419, Abcam) or mouse monoclonal anti-CD8 and rabbit polyclonal anti-CHOP/GADD153 (1:500, R-20, #sc-793, Santa Cruz Biotechnologies), followed by washes in PBS and incubation in secondary goat anti-mouse IgG1 and IgG2b or goat anti-mouse IgG1 and anti-rabbit IgG labeled with Alexa Fluor® 488 and 647 (all 1:200, ThermoFisher Scientific), respectively.

    Techniques: Expressing, Quantitation Assay, Activation Assay, Infection, Binding Assay, Chromatin Immunoprecipitation, Activity Assay, Real-time Polymerase Chain Reaction, Luciferase, Transfection, Fluorescence, FACS, Transduction

    CRPS patient serum and IgM had pronociceptive effects in B cell deficient fracture mice. At 3 weeks after tibia fracture and casting (FX) muMT mice lacking B cells and IgM exhibited unilateral hindpaw von Frey allodynia ( A ) and unweighting ( B ). After intraperitoneal injection of early (1–12 months post injury) CRPS patient serum (0.5ml, I.P) or IgM (500ug/1ml, I.P.) into 3 weeks post FX muMT mice, the mice gradually developed increased allodynia and unweighting over the ensuing week, and consistent with the 6 day half-life of IgM, these pronociceptive effects resolved by 2 weeks post-injection. The pronociceptive effects of the CRPS serum were restricted to the FX limb and there was no serum effect on post FX hindpaw edema or warmth (data not shown). No pronociceptive effects were observed after intraperitoneal injection of early CRPS patient IgG (5mg/1ml, I.P.) or after injection of control subject serum (0.5ml, I.P.) in 3 weeks post-FX mice. Measurements for ( A ) represent the difference between the FX side and contralateral paw, thus a negative value represents a decrease in mechanical withdrawal thresholds on the affected side. Measurements for ( B ) represent weight-bearing on the FX hind limb as a ratio to half of the total bilateral hind limb loading, thus, a percentage lower than 100% represents hindpaw unweighting. A 2-way repeated measures analysis of variance was used to test the effects of each treatment group on the dependent variables over time, using a Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SEM, n = 5 patients per cohort and each patients serum or immunoglobulin was injected into 3 mice for a total n of 15 mice. #P

    Journal: Pain

    Article Title: Complex regional pain syndrome patient IgM has pronociceptive effects in the skin and spinal cord of tibia fracture mice

    doi: 10.1097/j.pain.0000000000001765

    Figure Lengend Snippet: CRPS patient serum and IgM had pronociceptive effects in B cell deficient fracture mice. At 3 weeks after tibia fracture and casting (FX) muMT mice lacking B cells and IgM exhibited unilateral hindpaw von Frey allodynia ( A ) and unweighting ( B ). After intraperitoneal injection of early (1–12 months post injury) CRPS patient serum (0.5ml, I.P) or IgM (500ug/1ml, I.P.) into 3 weeks post FX muMT mice, the mice gradually developed increased allodynia and unweighting over the ensuing week, and consistent with the 6 day half-life of IgM, these pronociceptive effects resolved by 2 weeks post-injection. The pronociceptive effects of the CRPS serum were restricted to the FX limb and there was no serum effect on post FX hindpaw edema or warmth (data not shown). No pronociceptive effects were observed after intraperitoneal injection of early CRPS patient IgG (5mg/1ml, I.P.) or after injection of control subject serum (0.5ml, I.P.) in 3 weeks post-FX mice. Measurements for ( A ) represent the difference between the FX side and contralateral paw, thus a negative value represents a decrease in mechanical withdrawal thresholds on the affected side. Measurements for ( B ) represent weight-bearing on the FX hind limb as a ratio to half of the total bilateral hind limb loading, thus, a percentage lower than 100% represents hindpaw unweighting. A 2-way repeated measures analysis of variance was used to test the effects of each treatment group on the dependent variables over time, using a Sidak correction test for post hoc contrasts. Data are expressed as mean values ± SEM, n = 5 patients per cohort and each patients serum or immunoglobulin was injected into 3 mice for a total n of 15 mice. #P

    Article Snippet: [ ] IgG was extracted from CRPS serum using a POROS CaptureSelect™ IgG Affinity Matrix (Thermo Fisher Scientific, Leiden, Netherlands) with the pH adjusted to 7.4 using 1M Tris pH 8.0, and then Slide-A-Lyzer Dialysis Cassettes (10K MWCO, Life Technologies, Carlsbad, CA) were used to remove glycine from protein, and IgG quantified using a NanoDrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE).

    Techniques: Mouse Assay, Injection

    A Titer of BmVAL-1 specific IgG antibodies A ) and BmALT-2 specific IgG antibodies B ) in the sera of human subjects. Titer of antigen specific IgG antibodies were determined in sera samples from microfilaremic (n = 20), chronic pathology (n = 20) and endemic normal (NEN) (n = 20) subjects using an ELISA. Sera samples from NEN subjects served as controls. Each spot represents sera samples from one individual. The cut off value (mean OD ± 3 times SD of NEN sera) is indicated by a line drawn parallel to x-axis. B Titer of IgG isotype specific antibodies against rBmVAL-1 C ) and rBmALT-2 D ) in the sera samples of endemic normal subjects. Values were determined by specific ELISA for each isotype. Each spot represent sera samples from one individual. The IgG isotype antibody patterns against BmVAL-1 and BmALT-2 were comparable in the sera samples; N = 20.

    Journal: Research and reports in tropical medicine

    Article Title: Multivalent vaccine formulation with BmVAL-1 and BmALT-2 confer significant protection against challenge infections with Brugia malayi in mice and jirds

    doi: 10.2147/RRTM.S13679

    Figure Lengend Snippet: A Titer of BmVAL-1 specific IgG antibodies A ) and BmALT-2 specific IgG antibodies B ) in the sera of human subjects. Titer of antigen specific IgG antibodies were determined in sera samples from microfilaremic (n = 20), chronic pathology (n = 20) and endemic normal (NEN) (n = 20) subjects using an ELISA. Sera samples from NEN subjects served as controls. Each spot represents sera samples from one individual. The cut off value (mean OD ± 3 times SD of NEN sera) is indicated by a line drawn parallel to x-axis. B Titer of IgG isotype specific antibodies against rBmVAL-1 C ) and rBmALT-2 D ) in the sera samples of endemic normal subjects. Values were determined by specific ELISA for each isotype. Each spot represent sera samples from one individual. The IgG isotype antibody patterns against BmVAL-1 and BmALT-2 were comparable in the sera samples; N = 20.

    Article Snippet: Anti-BmVAL-1 and anti-BmALT-2 specific IgG1, IgG2a, IgG2b, IgG3, and IgG4 antibodies were determined in the sera of mouse using a mouse antibody isotyping kit purchased from Thermo Fisher Scientific.

    Techniques: Enzyme-linked Immunosorbent Assay

    Titer of anti-VAL-1 IgG and anti-ALT-2 IgG antibodies in the sera of immunized mice after 4 immunizations at 2-week intervals. IgG levels were measured using an ELISA. DNA – DNA vaccinated group, Protein – Protein vaccinated group, Prime boost – DNA Prime protein boost vaccinated group, and Control – vector plus alum controls. A) Sera collected from mice immunized with monovalent BmVAL-1 vaccine. B) Sera collected from mice immunized with monovalent BmALT-2 vaccine. C) Sera collected from mice immunized with BmVAL-1/BmALT-2 multivalent vaccine. Dotted lines: values for anti-BmVAL-1 IgG; solid lines: values for anti-BmALT-2 IgG; N = 10. Data represent results from one of two experiments with comparable results.

    Journal: Research and reports in tropical medicine

    Article Title: Multivalent vaccine formulation with BmVAL-1 and BmALT-2 confer significant protection against challenge infections with Brugia malayi in mice and jirds

    doi: 10.2147/RRTM.S13679

    Figure Lengend Snippet: Titer of anti-VAL-1 IgG and anti-ALT-2 IgG antibodies in the sera of immunized mice after 4 immunizations at 2-week intervals. IgG levels were measured using an ELISA. DNA – DNA vaccinated group, Protein – Protein vaccinated group, Prime boost – DNA Prime protein boost vaccinated group, and Control – vector plus alum controls. A) Sera collected from mice immunized with monovalent BmVAL-1 vaccine. B) Sera collected from mice immunized with monovalent BmALT-2 vaccine. C) Sera collected from mice immunized with BmVAL-1/BmALT-2 multivalent vaccine. Dotted lines: values for anti-BmVAL-1 IgG; solid lines: values for anti-BmALT-2 IgG; N = 10. Data represent results from one of two experiments with comparable results.

    Article Snippet: Anti-BmVAL-1 and anti-BmALT-2 specific IgG1, IgG2a, IgG2b, IgG3, and IgG4 antibodies were determined in the sera of mouse using a mouse antibody isotyping kit purchased from Thermo Fisher Scientific.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

    Levels of antiVAL-1 and anti-ALT-2 IgG isotype of antibodies were measured in the sera of immunized mice 2 weeks after the last immunization. A ) Monovalent BmVAL-1 vaccinated mice, B ) Monovalent BmALT-2 vaccinated mice, and C ) multivalent vaccinated mice; N = 10. Note : *Significant P

    Journal: Research and reports in tropical medicine

    Article Title: Multivalent vaccine formulation with BmVAL-1 and BmALT-2 confer significant protection against challenge infections with Brugia malayi in mice and jirds

    doi: 10.2147/RRTM.S13679

    Figure Lengend Snippet: Levels of antiVAL-1 and anti-ALT-2 IgG isotype of antibodies were measured in the sera of immunized mice 2 weeks after the last immunization. A ) Monovalent BmVAL-1 vaccinated mice, B ) Monovalent BmALT-2 vaccinated mice, and C ) multivalent vaccinated mice; N = 10. Note : *Significant P

    Article Snippet: Anti-BmVAL-1 and anti-BmALT-2 specific IgG1, IgG2a, IgG2b, IgG3, and IgG4 antibodies were determined in the sera of mouse using a mouse antibody isotyping kit purchased from Thermo Fisher Scientific.

    Techniques: Mouse Assay