igg1  (BioLegend)

 
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    Name:
    Ultra LEAF Purified Human IgG1 Isotype Control Recombinant
    Description:
    Ultra LEAF Purified Human IgG1 Isotype Control Recombinant QA16A12 Isotype Human IgG1 κ Reactivity Human Apps FC Size 100 μg
    Catalog Number:
    403501
    Price:
    225
    Category:
    Isotype Control
    Source:
    Human
    Applications:
    FC
    Conjugate:
    ULEAF
    Size:
    100 μg
    Quantity:
    1
    Preparation:
    The Ultra LEAF Low Endotoxin Azide Free antibody was purified by affinity chromatography
    Buy from Supplier


    Structured Review

    BioLegend igg1
    Impaired antibody responses to TD antigens in Cd4-Cre Tg Dock8 fl/fl mice. ( A ) TNP-specific serum <t>IgG</t> levels on day 0 (unimmunized) and day 21 (immunized) from Dock8 –/– (left), Cd4-Cre Tg Dock8 fl/fl mice (right), and controls after immunization in the bilateral hocks with TNP-KLH. ( B ) NP-specific serum IgG, TNP-specific serum IgG, and OVA-specific serum IgG measured on day 0 and day 21 after immunization of Cd4-Cre Tg Dock8 fl/fl mice and controls with NP-OVA in the hocks, TNP-KLH i.p., or OVA i.p. A and B show data from 1 representative experiment of 2. n = 4–5 mice/group. Data are presented as mean ± SEM. * P
    Ultra LEAF Purified Human IgG1 Isotype Control Recombinant QA16A12 Isotype Human IgG1 κ Reactivity Human Apps FC Size 100 μg
    https://www.bioz.com/result/igg1/product/BioLegend
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    igg1 - by Bioz Stars, 2021-05
    97/100 stars

    Images

    1) Product Images from "DOCK8 is essential for LFA-1–dependent positioning of T follicular helper cells in germinal centers"

    Article Title: DOCK8 is essential for LFA-1–dependent positioning of T follicular helper cells in germinal centers

    Journal: JCI Insight

    doi: 10.1172/jci.insight.134508

    Impaired antibody responses to TD antigens in Cd4-Cre Tg Dock8 fl/fl mice. ( A ) TNP-specific serum IgG levels on day 0 (unimmunized) and day 21 (immunized) from Dock8 –/– (left), Cd4-Cre Tg Dock8 fl/fl mice (right), and controls after immunization in the bilateral hocks with TNP-KLH. ( B ) NP-specific serum IgG, TNP-specific serum IgG, and OVA-specific serum IgG measured on day 0 and day 21 after immunization of Cd4-Cre Tg Dock8 fl/fl mice and controls with NP-OVA in the hocks, TNP-KLH i.p., or OVA i.p. A and B show data from 1 representative experiment of 2. n = 4–5 mice/group. Data are presented as mean ± SEM. * P
    Figure Legend Snippet: Impaired antibody responses to TD antigens in Cd4-Cre Tg Dock8 fl/fl mice. ( A ) TNP-specific serum IgG levels on day 0 (unimmunized) and day 21 (immunized) from Dock8 –/– (left), Cd4-Cre Tg Dock8 fl/fl mice (right), and controls after immunization in the bilateral hocks with TNP-KLH. ( B ) NP-specific serum IgG, TNP-specific serum IgG, and OVA-specific serum IgG measured on day 0 and day 21 after immunization of Cd4-Cre Tg Dock8 fl/fl mice and controls with NP-OVA in the hocks, TNP-KLH i.p., or OVA i.p. A and B show data from 1 representative experiment of 2. n = 4–5 mice/group. Data are presented as mean ± SEM. * P

    Techniques Used: Mouse Assay

    Cd4-Cre Tg Dock8 fl/fl mice have a normal Tfh percentage and phenotype after immunization with TD antigen. Cd4-Cre Tg Dock8 fl/fl mice and controls were immunized with TNP-KLH in the hock. Draining LNs were analyzed 7 days after immunization. ( A ) Representative FACS plots, and percentages and numbers of CXCR5 + PD-1 + Tfh cells. Pooled results from 3 individual experiments; n = 9 mice/group. ( B ) Ratio of Tfr to Tfh cells. ( C ) MFI of CXCR5, PD-1, and ICOS expression by Tfh cells. ( D ) Intracellular BCL6 expression by Tfh cells. ( E ) MFI of CD150, CD84, and CD40L expression by Tfh cells (left). qPCR analysis of Sh2d1a mRNA expression in sorted Tfh cells (right). Results are expressed as fold increase in Sh2d1a mRNA/β2 microglobulin mRNA ratio relative to control. ( F ) Intracellular expression of IL-4 and IL-21 by Tfh cells stimulated for 4 hours with phorbol-12,13-dibutyrate and ionomycin. ( G ) Percentage of B–T cell conjugates of total CD4 + T cells isolated from Dock8 –/– OT II and WT OT II mice incubated for 3 hours with LPS-stimulated WT B cells pulsed with OVA 323–339 . ( H ) Sorted CD4 + ICOS + CXCR5 + CD25 – CD19 − Tfh cells from the draining LNs were incubated with CD19 + B cells sorted from the LNs of WT mice in the presence of soluble anti-CD3 and anti-IgM. Cell surface expression of GL7, GLUT1, and IgG1 by B cells after 6 days in culture is shown. Results in B – H are representative of 3 independent experiments. Data are presented as mean ± SEM. Student’s t test; * P
    Figure Legend Snippet: Cd4-Cre Tg Dock8 fl/fl mice have a normal Tfh percentage and phenotype after immunization with TD antigen. Cd4-Cre Tg Dock8 fl/fl mice and controls were immunized with TNP-KLH in the hock. Draining LNs were analyzed 7 days after immunization. ( A ) Representative FACS plots, and percentages and numbers of CXCR5 + PD-1 + Tfh cells. Pooled results from 3 individual experiments; n = 9 mice/group. ( B ) Ratio of Tfr to Tfh cells. ( C ) MFI of CXCR5, PD-1, and ICOS expression by Tfh cells. ( D ) Intracellular BCL6 expression by Tfh cells. ( E ) MFI of CD150, CD84, and CD40L expression by Tfh cells (left). qPCR analysis of Sh2d1a mRNA expression in sorted Tfh cells (right). Results are expressed as fold increase in Sh2d1a mRNA/β2 microglobulin mRNA ratio relative to control. ( F ) Intracellular expression of IL-4 and IL-21 by Tfh cells stimulated for 4 hours with phorbol-12,13-dibutyrate and ionomycin. ( G ) Percentage of B–T cell conjugates of total CD4 + T cells isolated from Dock8 –/– OT II and WT OT II mice incubated for 3 hours with LPS-stimulated WT B cells pulsed with OVA 323–339 . ( H ) Sorted CD4 + ICOS + CXCR5 + CD25 – CD19 − Tfh cells from the draining LNs were incubated with CD19 + B cells sorted from the LNs of WT mice in the presence of soluble anti-CD3 and anti-IgM. Cell surface expression of GL7, GLUT1, and IgG1 by B cells after 6 days in culture is shown. Results in B – H are representative of 3 independent experiments. Data are presented as mean ± SEM. Student’s t test; * P

    Techniques Used: Mouse Assay, FACS, Expressing, Real-time Polymerase Chain Reaction, Isolation, Incubation

    2) Product Images from "Myeloid Dendritic Cells Induce HIV-1 Latency in Non-proliferating CD4+ T Cells"

    Article Title: Myeloid Dendritic Cells Induce HIV-1 Latency in Non-proliferating CD4+ T Cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003799

    DC-T cell interactions. ( A ) Productive and latent infection was quantified in eFluor670-labelled resting memory CD4 + T cells that were cultured either alone or with sorted mDC in the presence of media alone (light grey), anti-IgG (grey) or anti-CD18 (dark grey) prior to infection, n = 5. ( B ) Latent infection was determined in eFluor670-labelled resting CD4 + T cells that were cultured alone, with mDC or alternatively with soluble ICAM-1-fc and anti-IgG-fc, n = 2. ( C ) Latent infection was determined in sorted eFluor hi EGFP − CD4 + T cells following stimulation with anti-CD3/CD28, that were cultured alone or with mDC that were added prior to infection or post-infection, n = 5. Columns represent the median of 5 experiments and error bars the interquartile range. *P
    Figure Legend Snippet: DC-T cell interactions. ( A ) Productive and latent infection was quantified in eFluor670-labelled resting memory CD4 + T cells that were cultured either alone or with sorted mDC in the presence of media alone (light grey), anti-IgG (grey) or anti-CD18 (dark grey) prior to infection, n = 5. ( B ) Latent infection was determined in eFluor670-labelled resting CD4 + T cells that were cultured alone, with mDC or alternatively with soluble ICAM-1-fc and anti-IgG-fc, n = 2. ( C ) Latent infection was determined in sorted eFluor hi EGFP − CD4 + T cells following stimulation with anti-CD3/CD28, that were cultured alone or with mDC that were added prior to infection or post-infection, n = 5. Columns represent the median of 5 experiments and error bars the interquartile range. *P

    Techniques Used: Infection, Cell Culture

    3) Product Images from "The E3 ligases Itch and WWP2 cooperate to limit TH2 differentiation by enhancing signaling through the TCR"

    Article Title: The E3 ligases Itch and WWP2 cooperate to limit TH2 differentiation by enhancing signaling through the TCR

    Journal: Nature immunology

    doi: 10.1038/s41590-018-0137-8

    Itch interacts with WWP2 through its third WW domain. a , Immunoblot analysis of HEK293T cells transfected with expression vectors for Xpress-tagged Itch and/or Myc-tagged WWP2 or not (above lanes), assessed after immunoprecipitation (IP) with anti-Xpress (α-Xpress) or no immunoprecipitation (Total lysate) (left margin); right margin, molecular size in kilodaltons (throughout). b , Immunoblot analysis of the interaction of endogenous Itch with WWP2 in Jurkat T cells, assessed after immunoprecipitation with anti-Itch or the control antibody IgG (above lanes; left) or no immunoprecipitation (right). c , Confocal microscopy of the intracellular localization of Xpress-tagged Itch and Myc-tagged WWP2 in NIH3T3 cells transfected to express those molecules. Scale bars, 20 μm. d , Full-length (WT) Itch and its deletion mutants lacking functional domains (left; ranges (left margin) indicate amino acids present in construct), and immunoblot analysis of the interaction of Itch and WWP2 in HEK293T cells co-transfected (above lanes) to express Myc-tagged WWP2 plus empty vector (EV) or Xpress-tagged full-length Itch or various deletion mutants (middle and right), assessed after immunoprecipitation with anti-Myc (middle) or no immunoprecipitation (right). Data are representative of two to four independent experiments.
    Figure Legend Snippet: Itch interacts with WWP2 through its third WW domain. a , Immunoblot analysis of HEK293T cells transfected with expression vectors for Xpress-tagged Itch and/or Myc-tagged WWP2 or not (above lanes), assessed after immunoprecipitation (IP) with anti-Xpress (α-Xpress) or no immunoprecipitation (Total lysate) (left margin); right margin, molecular size in kilodaltons (throughout). b , Immunoblot analysis of the interaction of endogenous Itch with WWP2 in Jurkat T cells, assessed after immunoprecipitation with anti-Itch or the control antibody IgG (above lanes; left) or no immunoprecipitation (right). c , Confocal microscopy of the intracellular localization of Xpress-tagged Itch and Myc-tagged WWP2 in NIH3T3 cells transfected to express those molecules. Scale bars, 20 μm. d , Full-length (WT) Itch and its deletion mutants lacking functional domains (left; ranges (left margin) indicate amino acids present in construct), and immunoblot analysis of the interaction of Itch and WWP2 in HEK293T cells co-transfected (above lanes) to express Myc-tagged WWP2 plus empty vector (EV) or Xpress-tagged full-length Itch or various deletion mutants (middle and right), assessed after immunoprecipitation with anti-Myc (middle) or no immunoprecipitation (right). Data are representative of two to four independent experiments.

    Techniques Used: Transfection, Expressing, Immunoprecipitation, Confocal Microscopy, Functional Assay, Construct, Plasmid Preparation

    Accelerated autoimmunity and inflammation in DKO mice. a , Body weight of 6-week-old male wild-type (WT) mice, Wwp2 −/− , Itch f/f Cd4 -Cre and Itch f/f Cd4 -Cre Wwp2 −/− (DKO) mice (key; n = 8 per group). b , ELISA of IL-6 in the serum of 6- to 8-week-old mice as in a (key; n = 9 per group). c , ELISA of antibodies to double-stranded DNA (Anti-dsDNA) in the serum of 6-week-old mice as in a (key; n = 7 per group), presented as optical density at 450 nm (OD 450 ). d , Lung sections from 6-week-old mice as in a (above images), stained with hematoxylin and eosin. Scale bars, 100 μm. e , Absolute number of cells in the lungs (left) or bronchoalveolar lavage fluid (BALF) (right) of wild-type or DKObm mice (key; n = 8 per group) at 6-8 weeks after cell transplantation. f , Frequency (left) and number (right) of eosinophils, macrophages, dendritic cells and neutrophils (horizontal axis) in the bronchoalveolar lavage fluid of mice as in e (key; n = 8 per group (eosinophils, macrophages and neutrophils) or n = 5 per group (dendritic cells)). g , ELISA of the immunoglobulin subclasses IgE, IgG1 and IgA in serum from 6-week-old wild-type mice ( n = 5), Wwp2 −/− mice ( n = 6), Itch f/f Cd4 -Cre mice ( n = 6) or DKO mice ( n = 6) as in a (key). Each symbol ( a - c , e - g ) represents an individual mouse; small horizontal lines ( c , e , g ) indicate the mean (±s.d.). NS, not significant ( P > 0.05); * P
    Figure Legend Snippet: Accelerated autoimmunity and inflammation in DKO mice. a , Body weight of 6-week-old male wild-type (WT) mice, Wwp2 −/− , Itch f/f Cd4 -Cre and Itch f/f Cd4 -Cre Wwp2 −/− (DKO) mice (key; n = 8 per group). b , ELISA of IL-6 in the serum of 6- to 8-week-old mice as in a (key; n = 9 per group). c , ELISA of antibodies to double-stranded DNA (Anti-dsDNA) in the serum of 6-week-old mice as in a (key; n = 7 per group), presented as optical density at 450 nm (OD 450 ). d , Lung sections from 6-week-old mice as in a (above images), stained with hematoxylin and eosin. Scale bars, 100 μm. e , Absolute number of cells in the lungs (left) or bronchoalveolar lavage fluid (BALF) (right) of wild-type or DKObm mice (key; n = 8 per group) at 6-8 weeks after cell transplantation. f , Frequency (left) and number (right) of eosinophils, macrophages, dendritic cells and neutrophils (horizontal axis) in the bronchoalveolar lavage fluid of mice as in e (key; n = 8 per group (eosinophils, macrophages and neutrophils) or n = 5 per group (dendritic cells)). g , ELISA of the immunoglobulin subclasses IgE, IgG1 and IgA in serum from 6-week-old wild-type mice ( n = 5), Wwp2 −/− mice ( n = 6), Itch f/f Cd4 -Cre mice ( n = 6) or DKO mice ( n = 6) as in a (key). Each symbol ( a - c , e - g ) represents an individual mouse; small horizontal lines ( c , e , g ) indicate the mean (±s.d.). NS, not significant ( P > 0.05); * P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining, Transplantation Assay

    SHP-1 ubiquitination regulates the function of Lck. a , Immunoblot analysis of SHP-1 and actin (loading control) in lysates of wild-type and DKO Jurkat T cells (above blots) treated for 0, 1, 3 or 6 h (above lanes) with 50 μg/ml of cycloheximide (CHX). b , Immunoblot analysis of lysates of Jurkat T cells expressing wild-type SHP-1 or 3KR–SHP-1 (above blots) and treated with cycloheximide as in a . c , Protein tyrosine-phosphatase (PTP) activity of 3KR–SHP-1 among proteins immunoprecipitated, with the control antibody IgG or anti-Flag (below plot), from lysates of Jurkat T cells transfected with empty vector or expression vector for FLAG-tagged wild-type SHP-1 or 3KR–SHP-1 (key), presented relative to the activity of wild-type-SHP-1 (bottom), and immunoblot analysis of such immunoprecipitates (above lanes) (top). d , Immunoblot analysis of the interaction of SHP-1 and Lck in lysates of HEK293T cells transfected with expression vectors for wild-type SHP-1 or 3KR–SHP-1 plus either Lck (top group) or ZAP70 (bottom group) (above lanes), assessed after immunoprecipitation with anti-SHP-1 or without immunoprecipitation (left margin). e , Immunoblot analysis of the interaction of SHP-1 and Lck in Jurkat T cells transfected with expression vectors for wild-type SHP-1 or 3KR–SHP-1 (above lanes) and left unstimulated or stimulated for 5 min with anti-CD3 (above blots), assessed after immunoprecipitation with anti-SHP-1 or without immunoprecipitation (left margin). f , Immunoblot analysis of Lck phosphorylated at Tyr397 (p-Lck(Y397)) or Tyr505 (p-Lck(Y505)) and total Lck in CD4 + T cells sorted from wild-type, Wwp2 −/− , Itch f/f Cd4 -Cre or DKO mice (above blots) and stimulated for 0, 5 or 15 min (above lanes) with anti-CD3 plus anti-CD28; numbers below lanes indicate the ratio of phosphorylated protein to total protein. g , Immunoblot analysis of phosphorylated and total signaling molecules (left margin), and FLAG (loading control), in lysates of Jurkat T cells expressing short hairpin RNA targeting SHP-1 (SHP-1 shRNA) together with FLAG-tagged wild-type SHP-1 or 3KR–SHP-1 (above blots) and stimulated for 0, 2 or 15 min (above lanes) with anti-CD3 (numbers below lanes, as in f ) (right blots), and immunoblot analysis of SHP-1 in Jurkat T cells expressing control (non-targeting) or SHP-1-targeting short hairpin RNA (above lanes) (left). h , ELISA of IL-4 in supernatants of GFP + (transduced) CD4 + T cells sorted from bone marrow chimeras reconstituted with bone marrow cells transduced with retroviral vector encoding green fluorescent protein (GFP) and either wild-type SHP-1 or 3KR–SHP-1 (horizontal access), then stimulated for 48 h with anti-CD3 plus anti-CD28. i , ELISA of IL-4 in supernatants of naive CD4 + T cells transduced to express wild-type SHP-1 or 3KR–SHP-1 (horizontal axis) under neutral conditions (top) or T H 2 conditions (bottom) and then allowed to ‘rest’ for 3 d, followed by sorting of GFP + (transduced) cells and re-stimulation for 24 h with anti-CD3 plus anti-CD28. Each symbol ( c , h , i ) represents an individual technical replicate ( c , i ) or mouse ( h ). * P
    Figure Legend Snippet: SHP-1 ubiquitination regulates the function of Lck. a , Immunoblot analysis of SHP-1 and actin (loading control) in lysates of wild-type and DKO Jurkat T cells (above blots) treated for 0, 1, 3 or 6 h (above lanes) with 50 μg/ml of cycloheximide (CHX). b , Immunoblot analysis of lysates of Jurkat T cells expressing wild-type SHP-1 or 3KR–SHP-1 (above blots) and treated with cycloheximide as in a . c , Protein tyrosine-phosphatase (PTP) activity of 3KR–SHP-1 among proteins immunoprecipitated, with the control antibody IgG or anti-Flag (below plot), from lysates of Jurkat T cells transfected with empty vector or expression vector for FLAG-tagged wild-type SHP-1 or 3KR–SHP-1 (key), presented relative to the activity of wild-type-SHP-1 (bottom), and immunoblot analysis of such immunoprecipitates (above lanes) (top). d , Immunoblot analysis of the interaction of SHP-1 and Lck in lysates of HEK293T cells transfected with expression vectors for wild-type SHP-1 or 3KR–SHP-1 plus either Lck (top group) or ZAP70 (bottom group) (above lanes), assessed after immunoprecipitation with anti-SHP-1 or without immunoprecipitation (left margin). e , Immunoblot analysis of the interaction of SHP-1 and Lck in Jurkat T cells transfected with expression vectors for wild-type SHP-1 or 3KR–SHP-1 (above lanes) and left unstimulated or stimulated for 5 min with anti-CD3 (above blots), assessed after immunoprecipitation with anti-SHP-1 or without immunoprecipitation (left margin). f , Immunoblot analysis of Lck phosphorylated at Tyr397 (p-Lck(Y397)) or Tyr505 (p-Lck(Y505)) and total Lck in CD4 + T cells sorted from wild-type, Wwp2 −/− , Itch f/f Cd4 -Cre or DKO mice (above blots) and stimulated for 0, 5 or 15 min (above lanes) with anti-CD3 plus anti-CD28; numbers below lanes indicate the ratio of phosphorylated protein to total protein. g , Immunoblot analysis of phosphorylated and total signaling molecules (left margin), and FLAG (loading control), in lysates of Jurkat T cells expressing short hairpin RNA targeting SHP-1 (SHP-1 shRNA) together with FLAG-tagged wild-type SHP-1 or 3KR–SHP-1 (above blots) and stimulated for 0, 2 or 15 min (above lanes) with anti-CD3 (numbers below lanes, as in f ) (right blots), and immunoblot analysis of SHP-1 in Jurkat T cells expressing control (non-targeting) or SHP-1-targeting short hairpin RNA (above lanes) (left). h , ELISA of IL-4 in supernatants of GFP + (transduced) CD4 + T cells sorted from bone marrow chimeras reconstituted with bone marrow cells transduced with retroviral vector encoding green fluorescent protein (GFP) and either wild-type SHP-1 or 3KR–SHP-1 (horizontal access), then stimulated for 48 h with anti-CD3 plus anti-CD28. i , ELISA of IL-4 in supernatants of naive CD4 + T cells transduced to express wild-type SHP-1 or 3KR–SHP-1 (horizontal axis) under neutral conditions (top) or T H 2 conditions (bottom) and then allowed to ‘rest’ for 3 d, followed by sorting of GFP + (transduced) cells and re-stimulation for 24 h with anti-CD3 plus anti-CD28. Each symbol ( c , h , i ) represents an individual technical replicate ( c , i ) or mouse ( h ). * P

    Techniques Used: Expressing, Activity Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Mouse Assay, shRNA, Enzyme-linked Immunosorbent Assay, Transduction

    Itch and WWP2 promote the polyubiquitination of SHP-1. a , b , Immunoblot analysis of lysates of Jurkat T cells transfected to express FLAG-tagged Itch ( a ) or WWP2 ( b ), assessed after immunoprecipitation with anti-FLAG or the control antibody IgG (above lanes, left) or without immunoprecipitation (right). c , d , Immunoblot analysis of an in vivo ubiquitination assay of SHP-1 in HEK293T cells transfected with various combinations (above lanes) of empty vector and expression vectors for Xpress-tagged Itch, Myc-tagged WWP2, FLAG-tagged SHP-1 and hemagglutinin-tagged ubiquitin (HA-Ub) ( c ) or with expression vectors for Xpress-tagged Itch or FLAG-tagged WWP2 (each wild-type (WT) or catalytically inactive (with the active-site cysteine replaced with alanine (CA)), and hemagglutinin-tagged ubiquitin and Myc-tagged SHP-1 ( d ), then lysed under denaturing conditions and immunoprecipitated with anti-FLAG ( c ) or anti-Myc ( d ) or assessed without immunoprecipitation (left margin). e , Immunoblot analysis of the ubiquitination of SHP-1 in lysates of wild-type and DKO Jurkat T cells (above blots) transfected to express FLAG-tagged SHP-1 together with HA-tagged ubiquitin, stimulated for 0, 5 or 15 min (above lanes) with anti-CD3, assessed after immunoprecipitation with anti-FLAG (left margin). Data are representative of three to four independent experiments.
    Figure Legend Snippet: Itch and WWP2 promote the polyubiquitination of SHP-1. a , b , Immunoblot analysis of lysates of Jurkat T cells transfected to express FLAG-tagged Itch ( a ) or WWP2 ( b ), assessed after immunoprecipitation with anti-FLAG or the control antibody IgG (above lanes, left) or without immunoprecipitation (right). c , d , Immunoblot analysis of an in vivo ubiquitination assay of SHP-1 in HEK293T cells transfected with various combinations (above lanes) of empty vector and expression vectors for Xpress-tagged Itch, Myc-tagged WWP2, FLAG-tagged SHP-1 and hemagglutinin-tagged ubiquitin (HA-Ub) ( c ) or with expression vectors for Xpress-tagged Itch or FLAG-tagged WWP2 (each wild-type (WT) or catalytically inactive (with the active-site cysteine replaced with alanine (CA)), and hemagglutinin-tagged ubiquitin and Myc-tagged SHP-1 ( d ), then lysed under denaturing conditions and immunoprecipitated with anti-FLAG ( c ) or anti-Myc ( d ) or assessed without immunoprecipitation (left margin). e , Immunoblot analysis of the ubiquitination of SHP-1 in lysates of wild-type and DKO Jurkat T cells (above blots) transfected to express FLAG-tagged SHP-1 together with HA-tagged ubiquitin, stimulated for 0, 5 or 15 min (above lanes) with anti-CD3, assessed after immunoprecipitation with anti-FLAG (left margin). Data are representative of three to four independent experiments.

    Techniques Used: Transfection, Immunoprecipitation, In Vivo, Ubiquitin Assay, Plasmid Preparation, Expressing

    4) Product Images from "Deletion of WASp and N-WASp in B cells cripples the germinal center response and results in production of IgM autoantibodies"

    Article Title: Deletion of WASp and N-WASp in B cells cripples the germinal center response and results in production of IgM autoantibodies

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2015.06.003

    WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre/+ B cells can compete for help from WT T cells. (A) Generation of BM chimeric mice. WASp −/− or WASp −/− -WASp fl/fl CD19 Cre/+ (expressing CD45.2) and WT (CD45.1) BM cells at a 3:1 ratio were intravenous injected into lethally irradiated WT recipient mice. Injections of apoptotic cells started 10 weeks after transplantation. Percentage of cells was analyzed by flow cytometry. (B) Follicular B cells (B220 + CD23 + IgM int CD21 int ), MZB cells (B220 + CD23 − IgM high CD21 high ), total GC cells (B220 + GL7 + CD95 + ), LZ B cells (B220 + GL7 + CD95 + CD83 + CXCR4 low ), DZ B cells (B220 + GL7 + CD95 + CD83 low CXCR4 + ), total plasma cells (B220 − CD138 + ) and switched plasma cells (B220 − CD138 + IgG1 + ) were investigated at d 27. n = 7 per condition. (C) T FH cells (CD4 + CD44 + CD62L − PD1 + CXCR5 + ) were analyzed at d 27. n = 7 per condition. (D) B cell induced T cell activation in vitro . B cells were loaded with ovalbumin (OVA) and thereafter co-cultured with antigen-specific CD4 + T cells from OT-II mice. (E) Synapse formation between OVA-loaded B cells and OT-II CD4 + T cells quantified by immunohistochemistry. The star indicates the T cell and the arrow indicates a polarized synapse as determined by polymerized actin (F-actin) at the synapse interphase. Representative pictures from each strain from two experiments are shown. Bar, 10 μm n = 3. (F) Proliferation of and IL-2 production by CD4 + T cells from OT-II mice was measured by flow cytometry. n = 3. Graphs show three technical replicas and the experiment has been repeated five times with similar results. Abbreviations: WKO; WASp −/− , cDKO; WASp −/− -N-WASp fl/fl CD19 Cre+ .
    Figure Legend Snippet: WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre/+ B cells can compete for help from WT T cells. (A) Generation of BM chimeric mice. WASp −/− or WASp −/− -WASp fl/fl CD19 Cre/+ (expressing CD45.2) and WT (CD45.1) BM cells at a 3:1 ratio were intravenous injected into lethally irradiated WT recipient mice. Injections of apoptotic cells started 10 weeks after transplantation. Percentage of cells was analyzed by flow cytometry. (B) Follicular B cells (B220 + CD23 + IgM int CD21 int ), MZB cells (B220 + CD23 − IgM high CD21 high ), total GC cells (B220 + GL7 + CD95 + ), LZ B cells (B220 + GL7 + CD95 + CD83 + CXCR4 low ), DZ B cells (B220 + GL7 + CD95 + CD83 low CXCR4 + ), total plasma cells (B220 − CD138 + ) and switched plasma cells (B220 − CD138 + IgG1 + ) were investigated at d 27. n = 7 per condition. (C) T FH cells (CD4 + CD44 + CD62L − PD1 + CXCR5 + ) were analyzed at d 27. n = 7 per condition. (D) B cell induced T cell activation in vitro . B cells were loaded with ovalbumin (OVA) and thereafter co-cultured with antigen-specific CD4 + T cells from OT-II mice. (E) Synapse formation between OVA-loaded B cells and OT-II CD4 + T cells quantified by immunohistochemistry. The star indicates the T cell and the arrow indicates a polarized synapse as determined by polymerized actin (F-actin) at the synapse interphase. Representative pictures from each strain from two experiments are shown. Bar, 10 μm n = 3. (F) Proliferation of and IL-2 production by CD4 + T cells from OT-II mice was measured by flow cytometry. n = 3. Graphs show three technical replicas and the experiment has been repeated five times with similar results. Abbreviations: WKO; WASp −/− , cDKO; WASp −/− -N-WASp fl/fl CD19 Cre+ .

    Techniques Used: Mouse Assay, Expressing, Injection, Irradiation, Transplantation Assay, Flow Cytometry, Cytometry, Activation Assay, In Vitro, Cell Culture, Immunohistochemistry

    Increased autoantibody production in naive WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre+ mice. Serum from naïve mice and mice immunized with apoptotic cells were screened for reactivity to 95 different autoantigens using autoantibody array. Autoantigens are sorted by ANOVA starting with lowest p-value at the top for (A) IgM autoantibodies and for (B) IgG autoantibodies. Serum from 7 to 9 mice were tested, data of individual mice are shown. Black color equals ⩽ 1-fold change as compared with average values in control serum (WT d 0). A yellow square indicates a ⩾10-fold increase of autoantibody titer and a black square no difference as compared with average values in control serum. All yellow nuances in between represent a value larger than 1 and smaller than 10. WT, WASp −/− , WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 7, WT, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 9. (C–D) Autoantibodies in 8–10 weeks old mice. Serum titers of anti-DNA and anti-chromatin (C) IgM and (D) IgG measured in 8–10 weeks old unchallenged mice. WT n = 3, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 7, each dot correspond to one mouse. Significance was assessed with unpaired, two-tailed Student t test. * P
    Figure Legend Snippet: Increased autoantibody production in naive WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre+ mice. Serum from naïve mice and mice immunized with apoptotic cells were screened for reactivity to 95 different autoantigens using autoantibody array. Autoantigens are sorted by ANOVA starting with lowest p-value at the top for (A) IgM autoantibodies and for (B) IgG autoantibodies. Serum from 7 to 9 mice were tested, data of individual mice are shown. Black color equals ⩽ 1-fold change as compared with average values in control serum (WT d 0). A yellow square indicates a ⩾10-fold increase of autoantibody titer and a black square no difference as compared with average values in control serum. All yellow nuances in between represent a value larger than 1 and smaller than 10. WT, WASp −/− , WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 7, WT, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 9. (C–D) Autoantibodies in 8–10 weeks old mice. Serum titers of anti-DNA and anti-chromatin (C) IgM and (D) IgG measured in 8–10 weeks old unchallenged mice. WT n = 3, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 7, each dot correspond to one mouse. Significance was assessed with unpaired, two-tailed Student t test. * P

    Techniques Used: Mouse Assay, Two Tailed Test

    WASp −/− N-WASpn fl/fl CD19 Cre+ mice have increased serum titers of DNA-specific IgM antibodies. (A and B) Confocal imaging analysis of spleen sections from mice 30 min after injection with CFSE-labeled apoptotic cells. Representative images of WT and WASp −/− n = 3, WASp −/− -N-WASpn fl/fl nCD19 Cre+ n = 4, are shown. (A) B cells were visualized with B220 (blue), metallophilic macrophages with CD169 (red), and apoptotic CFSE (green). Bars, 150 μm. (B) DCs were visualized with CD11c (red). White arrows indicate co-localization of CD11c + cells and apoptotic cells. Bars, 300 μm (C and D) Flow cytometry analysis of DC subsets in spleen 30 min after injection with CFSE-labeled apoptotic cells (C) Absolute number of CD11c + DCs, CD11c + DEC2015 + DCs, and CD11c + 33D1 + DCs. (D) DC co-localization of CFSE-labeled apoptotic cells shown for CD11c + DEC205 + and CD11c + 33D1 + DCs. WT n = 5, WASp −/− n = 5, WASp −/− N-WASpn fl/fl nCD19 Cre+ n = 6. (E) Mice were injected with apoptotic cells once a week for four weeks. Serum titers of anti-DNA (F) IgM and (G) IgG antibodies were measured at d 0–27. n = 11 –19 and represent a pool of two experiments, each dot correspond to one mouse. Significance was assessed with unpaired, two-tailed Student t test. * P
    Figure Legend Snippet: WASp −/− N-WASpn fl/fl CD19 Cre+ mice have increased serum titers of DNA-specific IgM antibodies. (A and B) Confocal imaging analysis of spleen sections from mice 30 min after injection with CFSE-labeled apoptotic cells. Representative images of WT and WASp −/− n = 3, WASp −/− -N-WASpn fl/fl nCD19 Cre+ n = 4, are shown. (A) B cells were visualized with B220 (blue), metallophilic macrophages with CD169 (red), and apoptotic CFSE (green). Bars, 150 μm. (B) DCs were visualized with CD11c (red). White arrows indicate co-localization of CD11c + cells and apoptotic cells. Bars, 300 μm (C and D) Flow cytometry analysis of DC subsets in spleen 30 min after injection with CFSE-labeled apoptotic cells (C) Absolute number of CD11c + DCs, CD11c + DEC2015 + DCs, and CD11c + 33D1 + DCs. (D) DC co-localization of CFSE-labeled apoptotic cells shown for CD11c + DEC205 + and CD11c + 33D1 + DCs. WT n = 5, WASp −/− n = 5, WASp −/− N-WASpn fl/fl nCD19 Cre+ n = 6. (E) Mice were injected with apoptotic cells once a week for four weeks. Serum titers of anti-DNA (F) IgM and (G) IgG antibodies were measured at d 0–27. n = 11 –19 and represent a pool of two experiments, each dot correspond to one mouse. Significance was assessed with unpaired, two-tailed Student t test. * P

    Techniques Used: Mouse Assay, Imaging, Injection, Labeling, Flow Cytometry, Cytometry, Two Tailed Test

    Repeated apoptotic cell injections induce a GC response in WT, WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre/+ mice. (A) Immunohistochemistry of GC formation in spleen at d 0 and d 27 after apoptotic cell injections. B cells were labeled with B220 (blue), GC cells with PNA (red), and metallophilic macrophages with CD169 (green). Representative pictures from each strain from three experiments are shown. The ratio of GC area of total B220 + area in spleen sections is indicated. WT d 0 n = 8, WT d 27 n = 16, WASp −/− d 0 n = 7, WASp −/− d 27 n = 17, WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 8, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 16. Bars, 300 μm. (B) Flow cytometry data using B220, GL7 and CD95 to determine quantity of GC B cells. WT n = 14, WASp −/− n = 13, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 11. (C) Absolute number of T FH cells (CD4 + CD44 + CD62L − PD1 + CXCR5 + ) analyzed by flow cytometry. WT n = 8, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 5. (D) Flow cytometry data of the GC compartments as determined by LZ B cells (CD83 + CXCR4 − ) and DZ B cells (CD83 − CXCR4 + ). WT n = 14, WASp −/− n = 13, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 11. (E) Immunohistochemistry for GC polarization. FDCs were labeled with CD35 (green) in spleens at d 27 from mice immunized with apoptotic cells. Upper panel shows CD35 alone and lower panel, CD35 together with PNA (red) and B220 (blue). Representative pictures from each strain from two experiments are shown. The percentage of CD35 + area of total PNA + or B220 + area in spleen sections is indicated. n = 3 and three images per mouse were analyzed. Bars, 300 μm. (F) Absolute number of total plasma cells (B220 − CD138 + ) and IgG1 + plasma cells as determined by flow cytometry. WT n = 8, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 5. (G) Immunohistochemistry of IgM and IgG1 localization in spleen at d 0 and d 27. WT d 0 n = 3, WT d 27 n = 4, WASp −/− d 0 n = 4, WASp −/− d 27 n = 6, WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 4, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 6. Bars, 300 μm. Significance was assessed with unpaired, two-tailed Student t test. ** P
    Figure Legend Snippet: Repeated apoptotic cell injections induce a GC response in WT, WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre/+ mice. (A) Immunohistochemistry of GC formation in spleen at d 0 and d 27 after apoptotic cell injections. B cells were labeled with B220 (blue), GC cells with PNA (red), and metallophilic macrophages with CD169 (green). Representative pictures from each strain from three experiments are shown. The ratio of GC area of total B220 + area in spleen sections is indicated. WT d 0 n = 8, WT d 27 n = 16, WASp −/− d 0 n = 7, WASp −/− d 27 n = 17, WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 8, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 16. Bars, 300 μm. (B) Flow cytometry data using B220, GL7 and CD95 to determine quantity of GC B cells. WT n = 14, WASp −/− n = 13, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 11. (C) Absolute number of T FH cells (CD4 + CD44 + CD62L − PD1 + CXCR5 + ) analyzed by flow cytometry. WT n = 8, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 5. (D) Flow cytometry data of the GC compartments as determined by LZ B cells (CD83 + CXCR4 − ) and DZ B cells (CD83 − CXCR4 + ). WT n = 14, WASp −/− n = 13, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 11. (E) Immunohistochemistry for GC polarization. FDCs were labeled with CD35 (green) in spleens at d 27 from mice immunized with apoptotic cells. Upper panel shows CD35 alone and lower panel, CD35 together with PNA (red) and B220 (blue). Representative pictures from each strain from two experiments are shown. The percentage of CD35 + area of total PNA + or B220 + area in spleen sections is indicated. n = 3 and three images per mouse were analyzed. Bars, 300 μm. (F) Absolute number of total plasma cells (B220 − CD138 + ) and IgG1 + plasma cells as determined by flow cytometry. WT n = 8, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 5. (G) Immunohistochemistry of IgM and IgG1 localization in spleen at d 0 and d 27. WT d 0 n = 3, WT d 27 n = 4, WASp −/− d 0 n = 4, WASp −/− d 27 n = 6, WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 4, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 6. Bars, 300 μm. Significance was assessed with unpaired, two-tailed Student t test. ** P

    Techniques Used: Mouse Assay, Immunohistochemistry, Labeling, Flow Cytometry, Cytometry, Two Tailed Test

    Altered B cell affinity maturation in WASp −/− N-WASp fl/fl CD19 Cre/+ mice. (A and B) Characterization of replacement mutations found in the V H 1-186.2 family of (A) IgM and (B) IgG1. Grey background highlights the CDR1, CDR2, and CDR3 regions. Pink arrowhead indicates the high affinity amino acid mutation W33L. Amount of unique clones analyzed; WT Cμ n = 34, WT Cγ1 n = 109, WKO Cμ n = 36, WKO Cγ1 n = 34, cDKO Cμ n = 80, cDKO Cγ1 n = 21. (C) Breakdown of peripheral tolerance inWASp deficiency. N-WASp activity in WASp-deficient B cells supports increased reactivity to self antigens. Abbreviations: WKO; WASp −/− , cDKO; WASp −/− N-WASp fl/fl CD19 Cre/+ .
    Figure Legend Snippet: Altered B cell affinity maturation in WASp −/− N-WASp fl/fl CD19 Cre/+ mice. (A and B) Characterization of replacement mutations found in the V H 1-186.2 family of (A) IgM and (B) IgG1. Grey background highlights the CDR1, CDR2, and CDR3 regions. Pink arrowhead indicates the high affinity amino acid mutation W33L. Amount of unique clones analyzed; WT Cμ n = 34, WT Cγ1 n = 109, WKO Cμ n = 36, WKO Cγ1 n = 34, cDKO Cμ n = 80, cDKO Cγ1 n = 21. (C) Breakdown of peripheral tolerance inWASp deficiency. N-WASp activity in WASp-deficient B cells supports increased reactivity to self antigens. Abbreviations: WKO; WASp −/− , cDKO; WASp −/− N-WASp fl/fl CD19 Cre/+ .

    Techniques Used: Mouse Assay, Mutagenesis, Clone Assay, Activity Assay

    5) Product Images from "Immune Responses to SARS-CoV-2 Infection in Hospitalized Pediatric and Adult Patients"

    Article Title: Immune Responses to SARS-CoV-2 Infection in Hospitalized Pediatric and Adult Patients

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.abd5487

    Spike-protein specific antibody titers in patient serum samples. Anti-SARS-CoV-2 spike protein total IgG (A), IgA (B), IgG1 (C) and IgG3 (D) were measured by ELISA at a 1:50 serum dilution. Data are presented as optical density units (ODU) in relation to the time serum was obtained after admission (n=90) for IgG (Spearman, r=0.35, p=0.0008), IgA (r=0.35, p=0.0008), IgG1 (r=0.31, p=0.01) and IgG3 (r=0.24, p=0.047). (E) Shown is the ratio of anti-SARS-CoV-2 Spike protein-specific IgG1 to IgG3 in 71 patients with detectable IgG (Group 1, n=23; Group 2, n=10; Group 3, n=21, and Group 4, n=17) (*p
    Figure Legend Snippet: Spike-protein specific antibody titers in patient serum samples. Anti-SARS-CoV-2 spike protein total IgG (A), IgA (B), IgG1 (C) and IgG3 (D) were measured by ELISA at a 1:50 serum dilution. Data are presented as optical density units (ODU) in relation to the time serum was obtained after admission (n=90) for IgG (Spearman, r=0.35, p=0.0008), IgA (r=0.35, p=0.0008), IgG1 (r=0.31, p=0.01) and IgG3 (r=0.24, p=0.047). (E) Shown is the ratio of anti-SARS-CoV-2 Spike protein-specific IgG1 to IgG3 in 71 patients with detectable IgG (Group 1, n=23; Group 2, n=10; Group 3, n=21, and Group 4, n=17) (*p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    6) Product Images from "Herbal Formula-3 ameliorates OVA-induced food allergy in mice may via modulating the gut microbiota"

    Article Title: Herbal Formula-3 ameliorates OVA-induced food allergy in mice may via modulating the gut microbiota

    Journal: American Journal of Translational Research

    doi:

    OVA-specific antibodies IgE, IgG1, and cytokines IL-4, IL-5, IL-13 in mice at the day 42. CK, n = 7; FA, n = 6; FAHerb, n = 7. Different groups were shown in different colours. Data represent the mean ± standard error. Two tailed t-test was used
    Figure Legend Snippet: OVA-specific antibodies IgE, IgG1, and cytokines IL-4, IL-5, IL-13 in mice at the day 42. CK, n = 7; FA, n = 6; FAHerb, n = 7. Different groups were shown in different colours. Data represent the mean ± standard error. Two tailed t-test was used

    Techniques Used: Mouse Assay, Two Tailed Test

    7) Product Images from "Deletion of WASp and N-WASp in B cells cripples the germinal center response and results in production of IgM autoantibodies"

    Article Title: Deletion of WASp and N-WASp in B cells cripples the germinal center response and results in production of IgM autoantibodies

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2015.06.003

    WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre/+ B cells can compete for help from WT T cells. (A) Generation of BM chimeric mice. WASp −/− or WASp −/− -WASp fl/fl CD19 Cre/+ (expressing CD45.2) and WT (CD45.1) BM cells at a 3:1 ratio were intravenous injected into lethally irradiated WT recipient mice. Injections of apoptotic cells started 10 weeks after transplantation. Percentage of cells was analyzed by flow cytometry. (B) Follicular B cells (B220 + CD23 + IgM int CD21 int ), MZB cells (B220 + CD23 − IgM high CD21 high ), total GC cells (B220 + GL7 + CD95 + ), LZ B cells (B220 + GL7 + CD95 + CD83 + CXCR4 low ), DZ B cells (B220 + GL7 + CD95 + CD83 low CXCR4 + ), total plasma cells (B220 − CD138 + ) and switched plasma cells (B220 − CD138 + IgG1 + ) were investigated at d 27. n = 7 per condition. (C) T FH cells (CD4 + CD44 + CD62L − PD1 + CXCR5 + ) were analyzed at d 27. n = 7 per condition. (D) B cell induced T cell activation in vitro . B cells were loaded with ovalbumin (OVA) and thereafter co-cultured with antigen-specific CD4 + T cells from OT-II mice. (E) Synapse formation between OVA-loaded B cells and OT-II CD4 + T cells quantified by immunohistochemistry. The star indicates the T cell and the arrow indicates a polarized synapse as determined by polymerized actin (F-actin) at the synapse interphase. Representative pictures from each strain from two experiments are shown. Bar, 10 μm n = 3. (F) Proliferation of and IL-2 production by CD4 + T cells from OT-II mice was measured by flow cytometry. n = 3. Graphs show three technical replicas and the experiment has been repeated five times with similar results. Abbreviations: WKO; WASp −/− , cDKO; WASp −/− -N-WASp fl/fl CD19 Cre+ .
    Figure Legend Snippet: WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre/+ B cells can compete for help from WT T cells. (A) Generation of BM chimeric mice. WASp −/− or WASp −/− -WASp fl/fl CD19 Cre/+ (expressing CD45.2) and WT (CD45.1) BM cells at a 3:1 ratio were intravenous injected into lethally irradiated WT recipient mice. Injections of apoptotic cells started 10 weeks after transplantation. Percentage of cells was analyzed by flow cytometry. (B) Follicular B cells (B220 + CD23 + IgM int CD21 int ), MZB cells (B220 + CD23 − IgM high CD21 high ), total GC cells (B220 + GL7 + CD95 + ), LZ B cells (B220 + GL7 + CD95 + CD83 + CXCR4 low ), DZ B cells (B220 + GL7 + CD95 + CD83 low CXCR4 + ), total plasma cells (B220 − CD138 + ) and switched plasma cells (B220 − CD138 + IgG1 + ) were investigated at d 27. n = 7 per condition. (C) T FH cells (CD4 + CD44 + CD62L − PD1 + CXCR5 + ) were analyzed at d 27. n = 7 per condition. (D) B cell induced T cell activation in vitro . B cells were loaded with ovalbumin (OVA) and thereafter co-cultured with antigen-specific CD4 + T cells from OT-II mice. (E) Synapse formation between OVA-loaded B cells and OT-II CD4 + T cells quantified by immunohistochemistry. The star indicates the T cell and the arrow indicates a polarized synapse as determined by polymerized actin (F-actin) at the synapse interphase. Representative pictures from each strain from two experiments are shown. Bar, 10 μm n = 3. (F) Proliferation of and IL-2 production by CD4 + T cells from OT-II mice was measured by flow cytometry. n = 3. Graphs show three technical replicas and the experiment has been repeated five times with similar results. Abbreviations: WKO; WASp −/− , cDKO; WASp −/− -N-WASp fl/fl CD19 Cre+ .

    Techniques Used: Mouse Assay, Expressing, Injection, Irradiation, Transplantation Assay, Flow Cytometry, Cytometry, Activation Assay, In Vitro, Cell Culture, Immunohistochemistry

    Increased autoantibody production in naive WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre+ mice. Serum from naïve mice and mice immunized with apoptotic cells were screened for reactivity to 95 different autoantigens using autoantibody array. Autoantigens are sorted by ANOVA starting with lowest p-value at the top for (A) IgM autoantibodies and for (B) IgG autoantibodies. Serum from 7 to 9 mice were tested, data of individual mice are shown. Black color equals ⩽ 1-fold change as compared with average values in control serum (WT d 0). A yellow square indicates a ⩾10-fold increase of autoantibody titer and a black square no difference as compared with average values in control serum. All yellow nuances in between represent a value larger than 1 and smaller than 10. WT, WASp −/− , WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 7, WT, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 9. (C–D) Autoantibodies in 8–10 weeks old mice. Serum titers of anti-DNA and anti-chromatin (C) IgM and (D) IgG measured in 8–10 weeks old unchallenged mice. WT n = 3, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 7, each dot correspond to one mouse. Significance was assessed with unpaired, two-tailed Student t test. * P
    Figure Legend Snippet: Increased autoantibody production in naive WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre+ mice. Serum from naïve mice and mice immunized with apoptotic cells were screened for reactivity to 95 different autoantigens using autoantibody array. Autoantigens are sorted by ANOVA starting with lowest p-value at the top for (A) IgM autoantibodies and for (B) IgG autoantibodies. Serum from 7 to 9 mice were tested, data of individual mice are shown. Black color equals ⩽ 1-fold change as compared with average values in control serum (WT d 0). A yellow square indicates a ⩾10-fold increase of autoantibody titer and a black square no difference as compared with average values in control serum. All yellow nuances in between represent a value larger than 1 and smaller than 10. WT, WASp −/− , WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 7, WT, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 9. (C–D) Autoantibodies in 8–10 weeks old mice. Serum titers of anti-DNA and anti-chromatin (C) IgM and (D) IgG measured in 8–10 weeks old unchallenged mice. WT n = 3, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 7, each dot correspond to one mouse. Significance was assessed with unpaired, two-tailed Student t test. * P

    Techniques Used: Mouse Assay, Two Tailed Test

    WASp −/− N-WASpn fl/fl CD19 Cre+ mice have increased serum titers of DNA-specific IgM antibodies. (A and B) Confocal imaging analysis of spleen sections from mice 30 min after injection with CFSE-labeled apoptotic cells. Representative images of WT and WASp −/− n = 3, WASp −/− -N-WASpn fl/fl nCD19 Cre+ n = 4, are shown. (A) B cells were visualized with B220 (blue), metallophilic macrophages with CD169 (red), and apoptotic CFSE (green). Bars, 150 μm. (B) DCs were visualized with CD11c (red). White arrows indicate co-localization of CD11c + cells and apoptotic cells. Bars, 300 μm (C and D) Flow cytometry analysis of DC subsets in spleen 30 min after injection with CFSE-labeled apoptotic cells (C) Absolute number of CD11c + DCs, CD11c + DEC2015 + DCs, and CD11c + 33D1 + DCs. (D) DC co-localization of CFSE-labeled apoptotic cells shown for CD11c + DEC205 + and CD11c + 33D1 + DCs. WT n = 5, WASp −/− n = 5, WASp −/− N-WASpn fl/fl nCD19 Cre+ n = 6. (E) Mice were injected with apoptotic cells once a week for four weeks. Serum titers of anti-DNA (F) IgM and (G) IgG antibodies were measured at d 0–27. n = 11 –19 and represent a pool of two experiments, each dot correspond to one mouse. Significance was assessed with unpaired, two-tailed Student t test. * P
    Figure Legend Snippet: WASp −/− N-WASpn fl/fl CD19 Cre+ mice have increased serum titers of DNA-specific IgM antibodies. (A and B) Confocal imaging analysis of spleen sections from mice 30 min after injection with CFSE-labeled apoptotic cells. Representative images of WT and WASp −/− n = 3, WASp −/− -N-WASpn fl/fl nCD19 Cre+ n = 4, are shown. (A) B cells were visualized with B220 (blue), metallophilic macrophages with CD169 (red), and apoptotic CFSE (green). Bars, 150 μm. (B) DCs were visualized with CD11c (red). White arrows indicate co-localization of CD11c + cells and apoptotic cells. Bars, 300 μm (C and D) Flow cytometry analysis of DC subsets in spleen 30 min after injection with CFSE-labeled apoptotic cells (C) Absolute number of CD11c + DCs, CD11c + DEC2015 + DCs, and CD11c + 33D1 + DCs. (D) DC co-localization of CFSE-labeled apoptotic cells shown for CD11c + DEC205 + and CD11c + 33D1 + DCs. WT n = 5, WASp −/− n = 5, WASp −/− N-WASpn fl/fl nCD19 Cre+ n = 6. (E) Mice were injected with apoptotic cells once a week for four weeks. Serum titers of anti-DNA (F) IgM and (G) IgG antibodies were measured at d 0–27. n = 11 –19 and represent a pool of two experiments, each dot correspond to one mouse. Significance was assessed with unpaired, two-tailed Student t test. * P

    Techniques Used: Mouse Assay, Imaging, Injection, Labeling, Flow Cytometry, Cytometry, Two Tailed Test

    Repeated apoptotic cell injections induce a GC response in WT, WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre/+ mice. (A) Immunohistochemistry of GC formation in spleen at d 0 and d 27 after apoptotic cell injections. B cells were labeled with B220 (blue), GC cells with PNA (red), and metallophilic macrophages with CD169 (green). Representative pictures from each strain from three experiments are shown. The ratio of GC area of total B220 + area in spleen sections is indicated. WT d 0 n = 8, WT d 27 n = 16, WASp −/− d 0 n = 7, WASp −/− d 27 n = 17, WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 8, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 16. Bars, 300 μm. (B) Flow cytometry data using B220, GL7 and CD95 to determine quantity of GC B cells. WT n = 14, WASp −/− n = 13, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 11. (C) Absolute number of T FH cells (CD4 + CD44 + CD62L − PD1 + CXCR5 + ) analyzed by flow cytometry. WT n = 8, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 5. (D) Flow cytometry data of the GC compartments as determined by LZ B cells (CD83 + CXCR4 − ) and DZ B cells (CD83 − CXCR4 + ). WT n = 14, WASp −/− n = 13, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 11. (E) Immunohistochemistry for GC polarization. FDCs were labeled with CD35 (green) in spleens at d 27 from mice immunized with apoptotic cells. Upper panel shows CD35 alone and lower panel, CD35 together with PNA (red) and B220 (blue). Representative pictures from each strain from two experiments are shown. The percentage of CD35 + area of total PNA + or B220 + area in spleen sections is indicated. n = 3 and three images per mouse were analyzed. Bars, 300 μm. (F) Absolute number of total plasma cells (B220 − CD138 + ) and IgG1 + plasma cells as determined by flow cytometry. WT n = 8, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 5. (G) Immunohistochemistry of IgM and IgG1 localization in spleen at d 0 and d 27. WT d 0 n = 3, WT d 27 n = 4, WASp −/− d 0 n = 4, WASp −/− d 27 n = 6, WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 4, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 6. Bars, 300 μm. Significance was assessed with unpaired, two-tailed Student t test. ** P
    Figure Legend Snippet: Repeated apoptotic cell injections induce a GC response in WT, WASp −/− and WASp −/− N-WASp fl/fl CD19 Cre/+ mice. (A) Immunohistochemistry of GC formation in spleen at d 0 and d 27 after apoptotic cell injections. B cells were labeled with B220 (blue), GC cells with PNA (red), and metallophilic macrophages with CD169 (green). Representative pictures from each strain from three experiments are shown. The ratio of GC area of total B220 + area in spleen sections is indicated. WT d 0 n = 8, WT d 27 n = 16, WASp −/− d 0 n = 7, WASp −/− d 27 n = 17, WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 8, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 16. Bars, 300 μm. (B) Flow cytometry data using B220, GL7 and CD95 to determine quantity of GC B cells. WT n = 14, WASp −/− n = 13, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 11. (C) Absolute number of T FH cells (CD4 + CD44 + CD62L − PD1 + CXCR5 + ) analyzed by flow cytometry. WT n = 8, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 5. (D) Flow cytometry data of the GC compartments as determined by LZ B cells (CD83 + CXCR4 − ) and DZ B cells (CD83 − CXCR4 + ). WT n = 14, WASp −/− n = 13, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 11. (E) Immunohistochemistry for GC polarization. FDCs were labeled with CD35 (green) in spleens at d 27 from mice immunized with apoptotic cells. Upper panel shows CD35 alone and lower panel, CD35 together with PNA (red) and B220 (blue). Representative pictures from each strain from two experiments are shown. The percentage of CD35 + area of total PNA + or B220 + area in spleen sections is indicated. n = 3 and three images per mouse were analyzed. Bars, 300 μm. (F) Absolute number of total plasma cells (B220 − CD138 + ) and IgG1 + plasma cells as determined by flow cytometry. WT n = 8, WASp −/− n = 7, WASp −/− N-WASp fl/fl CD19 Cre/+ n = 5. (G) Immunohistochemistry of IgM and IgG1 localization in spleen at d 0 and d 27. WT d 0 n = 3, WT d 27 n = 4, WASp −/− d 0 n = 4, WASp −/− d 27 n = 6, WASp −/− N-WASp fl/fl CD19 Cre/+ d 0 n = 4, WASp −/− N-WASp fl/fl CD19 Cre/+ d 27 n = 6. Bars, 300 μm. Significance was assessed with unpaired, two-tailed Student t test. ** P

    Techniques Used: Mouse Assay, Immunohistochemistry, Labeling, Flow Cytometry, Cytometry, Two Tailed Test

    Altered B cell affinity maturation in WASp −/− N-WASp fl/fl CD19 Cre/+ mice. (A and B) Characterization of replacement mutations found in the V H 1-186.2 family of (A) IgM and (B) IgG1. Grey background highlights the CDR1, CDR2, and CDR3 regions. Pink arrowhead indicates the high affinity amino acid mutation W33L. Amount of unique clones analyzed; WT Cμ n = 34, WT Cγ1 n = 109, WKO Cμ n = 36, WKO Cγ1 n = 34, cDKO Cμ n = 80, cDKO Cγ1 n = 21. (C) Breakdown of peripheral tolerance inWASp deficiency. N-WASp activity in WASp-deficient B cells supports increased reactivity to self antigens. Abbreviations: WKO; WASp −/− , cDKO; WASp −/− N-WASp fl/fl CD19 Cre/+ .
    Figure Legend Snippet: Altered B cell affinity maturation in WASp −/− N-WASp fl/fl CD19 Cre/+ mice. (A and B) Characterization of replacement mutations found in the V H 1-186.2 family of (A) IgM and (B) IgG1. Grey background highlights the CDR1, CDR2, and CDR3 regions. Pink arrowhead indicates the high affinity amino acid mutation W33L. Amount of unique clones analyzed; WT Cμ n = 34, WT Cγ1 n = 109, WKO Cμ n = 36, WKO Cγ1 n = 34, cDKO Cμ n = 80, cDKO Cγ1 n = 21. (C) Breakdown of peripheral tolerance inWASp deficiency. N-WASp activity in WASp-deficient B cells supports increased reactivity to self antigens. Abbreviations: WKO; WASp −/− , cDKO; WASp −/− N-WASp fl/fl CD19 Cre/+ .

    Techniques Used: Mouse Assay, Mutagenesis, Clone Assay, Activity Assay

    8) Product Images from "Pentalinonsterol, a Constituent of Pentalinon andrieuxii, Possesses Potent Immunomodulatory Activity and Primes T Cell Immune Responses"

    Article Title: Pentalinonsterol, a Constituent of Pentalinon andrieuxii, Possesses Potent Immunomodulatory Activity and Primes T Cell Immune Responses

    Journal: Journal of natural products

    doi: 10.1021/acs.jnatprod.7b00445

    (A) Schema for mouse immunization experiments with PEN. Mice were immunized with OVA + 10 μ g of PEN, OVA + 100 μ g of PEN, or OVA + vehicle (control) at week 0 and boosted at weeks 2 and 4. (B, C) OVA-specific (B) IgG2a and (C) IgG1 antibody titers from serum of immunized mice in each group obtained at weeks 2, 4, 6, 8, and 10, as determined by ELISA. Data shown are the combined mean ± SEM values from three independent experiments ( n = 5 per group) (* represents a p value of
    Figure Legend Snippet: (A) Schema for mouse immunization experiments with PEN. Mice were immunized with OVA + 10 μ g of PEN, OVA + 100 μ g of PEN, or OVA + vehicle (control) at week 0 and boosted at weeks 2 and 4. (B, C) OVA-specific (B) IgG2a and (C) IgG1 antibody titers from serum of immunized mice in each group obtained at weeks 2, 4, 6, 8, and 10, as determined by ELISA. Data shown are the combined mean ± SEM values from three independent experiments ( n = 5 per group) (* represents a p value of

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    9) Product Images from "Immunologically programming the tumor microenvironment induces the pattern recognition receptor NLRC4-dependent antitumor immunity"

    Article Title: Immunologically programming the tumor microenvironment induces the pattern recognition receptor NLRC4-dependent antitumor immunity

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1136/jitc-2020-001595

    GM-CSF production induced by Flagrp170 immune programming is critical for its antitumor efficacy. (A) B16 tumor-bearing C57BL/6 mice (n=3) received in situ Flagrp170 therapy for a total of five doses. Tumor tissues were collected 24 hours after each treatment and subjected to analyzes of Th1 immunity-related genes. (B and C) Production of GM-CSF by tumor-infiltrating immune cell subsets was examined by intracellular cytokine staining and flow cytometry. (D) B16 tumor-bearing mice (n=3) were depleted of CD4 + or CD8 + T cells prior to Flagrp170 therapy by administration of the corresponding antibodies. Transcription of gmcsf gene in tumor tissues was assayed by quantitative PCR. Flagrp170-treated mice receiving normal IgG and mice receiving empty adenovirus serve as controls. (E) Neutralization of GM-CSF inhibits antitumor activity of Flagrp170. B16 tumor-bearing mice (n=5) were injected with GM-CSF neutralizing antibodies during in situ Flagrp170 treatment. (F) Tumor infiltration by CD11b + CD11c + dendritic cells was determined by flow cytometry. (G) Transcription of ifng, il12a , and gmcsf in tumor tissues (n=3) was assayed by quantitative PCR. Data shown are representative of three independent experiments. *p
    Figure Legend Snippet: GM-CSF production induced by Flagrp170 immune programming is critical for its antitumor efficacy. (A) B16 tumor-bearing C57BL/6 mice (n=3) received in situ Flagrp170 therapy for a total of five doses. Tumor tissues were collected 24 hours after each treatment and subjected to analyzes of Th1 immunity-related genes. (B and C) Production of GM-CSF by tumor-infiltrating immune cell subsets was examined by intracellular cytokine staining and flow cytometry. (D) B16 tumor-bearing mice (n=3) were depleted of CD4 + or CD8 + T cells prior to Flagrp170 therapy by administration of the corresponding antibodies. Transcription of gmcsf gene in tumor tissues was assayed by quantitative PCR. Flagrp170-treated mice receiving normal IgG and mice receiving empty adenovirus serve as controls. (E) Neutralization of GM-CSF inhibits antitumor activity of Flagrp170. B16 tumor-bearing mice (n=5) were injected with GM-CSF neutralizing antibodies during in situ Flagrp170 treatment. (F) Tumor infiltration by CD11b + CD11c + dendritic cells was determined by flow cytometry. (G) Transcription of ifng, il12a , and gmcsf in tumor tissues (n=3) was assayed by quantitative PCR. Data shown are representative of three independent experiments. *p

    Techniques Used: Mouse Assay, In Situ, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Neutralization, Activity Assay, Injection

    10) Product Images from "Toll-like receptor 9 antagonizes antibody affinity maturation"

    Article Title: Toll-like receptor 9 antagonizes antibody affinity maturation

    Journal: Nature immunology

    doi: 10.1038/s41590-018-0052-z

    CpG does not induce high affinity antibodies in human Human subjects were immunized with AMA1-C1, formulated on Alhydrogel with or without 564 μg of CpG 7909. Sera were collected on day 70 post immunization and IgG antibodies were purified using protein G columns. Representative chromatography profiles (a) are shown. (b) BIAcore binding analysis between PfAMA1 and serum IgGs (70nM). The binding sensorgrams of IgG samples from CpG and Non-CpG immunized subjects are shown in blue and red, respectively. (c,d) Apparent K D binding affinities (c) and k off values (d) measured by surface plasmon resonance analysis of serially diluted purified antibodies are graphed. Each symbol represents an individual. Statistical significance was measured using a two sided Welch’s t-test
    Figure Legend Snippet: CpG does not induce high affinity antibodies in human Human subjects were immunized with AMA1-C1, formulated on Alhydrogel with or without 564 μg of CpG 7909. Sera were collected on day 70 post immunization and IgG antibodies were purified using protein G columns. Representative chromatography profiles (a) are shown. (b) BIAcore binding analysis between PfAMA1 and serum IgGs (70nM). The binding sensorgrams of IgG samples from CpG and Non-CpG immunized subjects are shown in blue and red, respectively. (c,d) Apparent K D binding affinities (c) and k off values (d) measured by surface plasmon resonance analysis of serially diluted purified antibodies are graphed. Each symbol represents an individual. Statistical significance was measured using a two sided Welch’s t-test

    Techniques Used: Purification, Chromatography, Binding Assay, SPR Assay

    The B cell intrinsic expression of the TLR9 adaptor MyD88 impacts the outcome of T cell dependent Ab response in vivo ). (a–d) Representative flow plots (a), total number of GC B cells (b) , percent of all GC B cells that were NP-specific (c) and the total number of NP- specific GC B cells (d). (e–h) Representative flow plots (e) , total number of IgG + IgD − cells (f) , percent of all IgG + IgD − B cells that were NP-specific (g) and total number of NP-specific IgG + IgD − B cells (h). (i–l) Representative flow plots (i) and total number of PC-lineage cells (j) . NP-specific IgG Ab secreting cells (k) and visualized by ELISPOT (l). Data were pooled from two independent experiments. Each symbol represents an individual mouse. Dotted lines indicate mean values. Statistical significance was calculated using a two sided Welch’s t test. (m–o) NP-specific IgM (n) , NP-specific IgG (o) and high affinity NP-specific IgG (p) given in arbitrary units calculated from serial dilutions of pooled sera from NP-CGG immunized WT mice. Data represent two independent experiments. Lines and error bars represent the mean of 8 individual mice and SD respectively. A two sided Welch’s t test was used to determine the statistical significance. Values that are significantly different between each group for each collection day are shown with asterisks (n.s.= P > 0.05; *=0.01
    Figure Legend Snippet: The B cell intrinsic expression of the TLR9 adaptor MyD88 impacts the outcome of T cell dependent Ab response in vivo ). (a–d) Representative flow plots (a), total number of GC B cells (b) , percent of all GC B cells that were NP-specific (c) and the total number of NP- specific GC B cells (d). (e–h) Representative flow plots (e) , total number of IgG + IgD − cells (f) , percent of all IgG + IgD − B cells that were NP-specific (g) and total number of NP-specific IgG + IgD − B cells (h). (i–l) Representative flow plots (i) and total number of PC-lineage cells (j) . NP-specific IgG Ab secreting cells (k) and visualized by ELISPOT (l). Data were pooled from two independent experiments. Each symbol represents an individual mouse. Dotted lines indicate mean values. Statistical significance was calculated using a two sided Welch’s t test. (m–o) NP-specific IgM (n) , NP-specific IgG (o) and high affinity NP-specific IgG (p) given in arbitrary units calculated from serial dilutions of pooled sera from NP-CGG immunized WT mice. Data represent two independent experiments. Lines and error bars represent the mean of 8 individual mice and SD respectively. A two sided Welch’s t test was used to determine the statistical significance. Values that are significantly different between each group for each collection day are shown with asterisks (n.s.= P > 0.05; *=0.01

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

    The effect of TLR9 signaling on the outcome of B cell responses to antigen In all cases purified mouse splenic B cells (WT or TRL9 KO) were stimulated in vitro with Anti-IgM (2–5μg/ml) or CpG (1μM) alone or in combination. (a–d) , pooled, permeabilized and stained with mAbs specific for the phospho-kinases: p-Syk (a) , p-Btk (b) , p-p38 (c) and p-Akt (d) . The fold changes in abundance of phosphorylated kinases in stimulated as compared to unstimulated B cells are shown. (e) Calcium flux measured by flow cytometry in B cells loaded with the Ca 2+ sensor dyes Furo-red and Fluo-4 and stimulated. (f) Fold changes in the mRNA expression for various cytokines of B cells stimulated for 4h as compared to unstimulated B cells. ( g) ELISA measurements of cytokine proteins in the culture supernatants of WT or TLR9 KO B cells stimulated in vitro for 18 h (for IL-6) or 24 h (for TNF, IL-2 and IL-10). (h) Proliferation of WT or TLR9 KO B cells stimulated with a sub-optimal concentration of Anti-IgM (1μg/ml) and increasing concentrations of CpG (0 to 3 μM). Shown are the percentage of cells that proliferated after 46 h of culture. (i,j) Antibody production by stimulated B cells for a duration of seven days. ELISA measurement of IgM (i) and IgG from the IgG + ) (j). (k–m) Kinetic analysis of in vitro mRNA expression of GC B cell- or PC-specific genes in stimulated WT B cells for 4 days. Expression of Bcl6 ( k ), Prdm1 ( l ) and Aicda ( m ) is shown as fold changes over that observed in unstimulated B cells at time 0. Data are representative of three independent experiments performed with duplicate ( a–d ), or triplicate samples ( e–n) . Data points and error bars indicate mean and standard deviation, respectively. Statistical significance was measured using two sided unpaired t-test (**= 0.001
    Figure Legend Snippet: The effect of TLR9 signaling on the outcome of B cell responses to antigen In all cases purified mouse splenic B cells (WT or TRL9 KO) were stimulated in vitro with Anti-IgM (2–5μg/ml) or CpG (1μM) alone or in combination. (a–d) , pooled, permeabilized and stained with mAbs specific for the phospho-kinases: p-Syk (a) , p-Btk (b) , p-p38 (c) and p-Akt (d) . The fold changes in abundance of phosphorylated kinases in stimulated as compared to unstimulated B cells are shown. (e) Calcium flux measured by flow cytometry in B cells loaded with the Ca 2+ sensor dyes Furo-red and Fluo-4 and stimulated. (f) Fold changes in the mRNA expression for various cytokines of B cells stimulated for 4h as compared to unstimulated B cells. ( g) ELISA measurements of cytokine proteins in the culture supernatants of WT or TLR9 KO B cells stimulated in vitro for 18 h (for IL-6) or 24 h (for TNF, IL-2 and IL-10). (h) Proliferation of WT or TLR9 KO B cells stimulated with a sub-optimal concentration of Anti-IgM (1μg/ml) and increasing concentrations of CpG (0 to 3 μM). Shown are the percentage of cells that proliferated after 46 h of culture. (i,j) Antibody production by stimulated B cells for a duration of seven days. ELISA measurement of IgM (i) and IgG from the IgG + ) (j). (k–m) Kinetic analysis of in vitro mRNA expression of GC B cell- or PC-specific genes in stimulated WT B cells for 4 days. Expression of Bcl6 ( k ), Prdm1 ( l ) and Aicda ( m ) is shown as fold changes over that observed in unstimulated B cells at time 0. Data are representative of three independent experiments performed with duplicate ( a–d ), or triplicate samples ( e–n) . Data points and error bars indicate mean and standard deviation, respectively. Statistical significance was measured using two sided unpaired t-test (**= 0.001

    Techniques Used: Purification, In Vitro, Staining, Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation

    11) Product Images from "Decreased expression of CD200R3 on mouse basophils as a novel marker for IgG1-mediated anaphylaxis"

    Article Title: Decreased expression of CD200R3 on mouse basophils as a novel marker for IgG1-mediated anaphylaxis

    Journal: Immunity, Inflammation and Disease

    doi: 10.1002/iid3.67

    Decreased basophilic surface CD200R3 expression after induction of IgG-mediated anaphylaxis. (a) Mice were injected intravenously with 2.4G2 or isotype control, and rectal temperatures were monitored. Four mice per injection group were used. (b) The mice were then bled before injection (0 h), and 0.5, 4, 8, and 24 h afterwards. Basophil CD200R3 expression was subsequently evaluated. Data are represented as means ± SDs ( n = 4). Asterisks indicate significant differences between the two groups at each time point ( *P
    Figure Legend Snippet: Decreased basophilic surface CD200R3 expression after induction of IgG-mediated anaphylaxis. (a) Mice were injected intravenously with 2.4G2 or isotype control, and rectal temperatures were monitored. Four mice per injection group were used. (b) The mice were then bled before injection (0 h), and 0.5, 4, 8, and 24 h afterwards. Basophil CD200R3 expression was subsequently evaluated. Data are represented as means ± SDs ( n = 4). Asterisks indicate significant differences between the two groups at each time point ( *P

    Techniques Used: Expressing, Mouse Assay, Injection

    Basophil CD200R3 expression following depletion of specific IgG subclasses from antiserum used for passive sensitization. Each IgG subclass was depleted from mouse anti-β-LG serum using biotinylated antibodies specific to IgG1, IgG2a, IgG2b, IgG3, or corresponding isotype controls. (a) IgG subclass-specific depletion from antiserum was confirmed by ELISA. (b) Mouse peripheral blood was then sensitized with various dilutions of the depleted antisera. Antigen challenge followed by flow cytometry analysis was carried out as described in Figure 4 . Significant differences compared to the isotype control group are indicated by asterisks (* P
    Figure Legend Snippet: Basophil CD200R3 expression following depletion of specific IgG subclasses from antiserum used for passive sensitization. Each IgG subclass was depleted from mouse anti-β-LG serum using biotinylated antibodies specific to IgG1, IgG2a, IgG2b, IgG3, or corresponding isotype controls. (a) IgG subclass-specific depletion from antiserum was confirmed by ELISA. (b) Mouse peripheral blood was then sensitized with various dilutions of the depleted antisera. Antigen challenge followed by flow cytometry analysis was carried out as described in Figure 4 . Significant differences compared to the isotype control group are indicated by asterisks (* P

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

    Basophil CD200R3 expression levels following IgG-specific depletion from antiserum used for passive sensitization. (a) Mouse antiserum was treated with either Protein G Sepharose 4FF or Sepharose 4B and β-LG-specific IgG were measured by ELISA. (b) Mouse peripheral blood was sensitized with antiserum treated with either Protein G Sepharose 4FF or Sepharose 4B, followed by antigen challenge with 1 μg/mL β-LG for 2 h. The expression level of CD200R3 was analyzed by flow cytometry and is given as relative expression level. Data are represented as means ± SDs ( n = 3). Significant differences between groups are indicated by asterisks ( *P
    Figure Legend Snippet: Basophil CD200R3 expression levels following IgG-specific depletion from antiserum used for passive sensitization. (a) Mouse antiserum was treated with either Protein G Sepharose 4FF or Sepharose 4B and β-LG-specific IgG were measured by ELISA. (b) Mouse peripheral blood was sensitized with antiserum treated with either Protein G Sepharose 4FF or Sepharose 4B, followed by antigen challenge with 1 μg/mL β-LG for 2 h. The expression level of CD200R3 was analyzed by flow cytometry and is given as relative expression level. Data are represented as means ± SDs ( n = 3). Significant differences between groups are indicated by asterisks ( *P

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

    Increased expression of CD200R1 and decreased expression of CD200R3 on basophils in response to IgE and IgG receptor-mediated activation, respectively. Peripheral blood was incubated at 37°C for 2 h with 100 ng/mL anti-mouse FcγRIII/II (2.4G2), 300 ng/mL goat anti-mouse IgE, or the respective isotype control antibody. Relative expression levels of CD200R1 and CD200R3 on basophils are shown. Data are represented as means ± SDs ( n = 3). Asterisks indicate significant differences ( P
    Figure Legend Snippet: Increased expression of CD200R1 and decreased expression of CD200R3 on basophils in response to IgE and IgG receptor-mediated activation, respectively. Peripheral blood was incubated at 37°C for 2 h with 100 ng/mL anti-mouse FcγRIII/II (2.4G2), 300 ng/mL goat anti-mouse IgE, or the respective isotype control antibody. Relative expression levels of CD200R1 and CD200R3 on basophils are shown. Data are represented as means ± SDs ( n = 3). Asterisks indicate significant differences ( P

    Techniques Used: Expressing, Activation Assay, Incubation

    12) Product Images from "Allergen-specific IgG antibodies signaling via FcγRIIb promote food tolerance"

    Article Title: Allergen-specific IgG antibodies signaling via FcγRIIb promote food tolerance

    Journal: The Journal of allergy and clinical immunology

    doi: 10.1016/j.jaci.2017.03.045

    Impact of mast cell FcγRIIb ligation on the development of Treg responses during sensitization. A) Anaphylaxis in Il4raF709 Kit W-sh mice on the C57BL/6 background reconstituted with WT or FcγRIIb −/− mast cells. n=3 F709 Kit W-sh No MC, n=5 WT MC, n=4 WT MC αOVA IgG, n=4 FcγRIIb −/− MC, n=5 FcγRIIb −/− MC αOVA IgG. B) Serum IL-4 after challenge. C) Serum OVA-specific IgE. n=6 F709 Kit W-sh No MC, n=8 WT MC, n=8 WT MC αOVA IgG, n=8 FcγRIIb −/− MC, n=9 FcγRIIb −/− MC αOVA IgG. D) Intestinal mast cells. E) Proportional representation of Treg in the small intestine. F) Intestinal Th2 frequencies. Data shown are from one of three independent experiments.
    Figure Legend Snippet: Impact of mast cell FcγRIIb ligation on the development of Treg responses during sensitization. A) Anaphylaxis in Il4raF709 Kit W-sh mice on the C57BL/6 background reconstituted with WT or FcγRIIb −/− mast cells. n=3 F709 Kit W-sh No MC, n=5 WT MC, n=4 WT MC αOVA IgG, n=4 FcγRIIb −/− MC, n=5 FcγRIIb −/− MC αOVA IgG. B) Serum IL-4 after challenge. C) Serum OVA-specific IgE. n=6 F709 Kit W-sh No MC, n=8 WT MC, n=8 WT MC αOVA IgG, n=8 FcγRIIb −/− MC, n=9 FcγRIIb −/− MC αOVA IgG. D) Intestinal mast cells. E) Proportional representation of Treg in the small intestine. F) Intestinal Th2 frequencies. Data shown are from one of three independent experiments.

    Techniques Used: Ligation, Mouse Assay

    Impact of exogenous IgG on allergen desensitization. A) Anaphylaxis in OVA-sensitized and desensitized BALB/c background Il4raF709 mice after challenge. n=4 Sensitized only, n=5 OVA + control IgG, and n=4 OVA + αOVA IgG. B) MMCP-1 release. C) IL-4 release. D) Intestinal mast cell numbers. E) OVA-specific IgE levels. F) Intestinal Treg frequencies. G) Intestinal Th2 frequencies. Data shown are representative of three independent experiments.
    Figure Legend Snippet: Impact of exogenous IgG on allergen desensitization. A) Anaphylaxis in OVA-sensitized and desensitized BALB/c background Il4raF709 mice after challenge. n=4 Sensitized only, n=5 OVA + control IgG, and n=4 OVA + αOVA IgG. B) MMCP-1 release. C) IL-4 release. D) Intestinal mast cell numbers. E) OVA-specific IgE levels. F) Intestinal Treg frequencies. G) Intestinal Th2 frequencies. Data shown are representative of three independent experiments.

    Techniques Used: Mouse Assay

    Role of IgG and FcγRIIb and in tolerance restoration during desensitization. A) Anaphylaxis following enteral challenge in OVA-sensitized and desensitized Il4raF709 and Il4raF709 FcγRIIb −/− mice on the C57BL/6 background. ns, not significantly different. B) Serum levels IL-4. C) Intestinal mast cells. D) Specific IgE levels. E) Intestinal Treg frequencies. F) Intestinal Th2 frequencies. n=4–5 WT Control IgG, n=3–4 WT αOVA IgG, n=4–5 FcγRIIb −/− Control IgG and n=4–5 FcγRIIb −/− αOVA IgG. Data shown are from one of two independent experiments.
    Figure Legend Snippet: Role of IgG and FcγRIIb and in tolerance restoration during desensitization. A) Anaphylaxis following enteral challenge in OVA-sensitized and desensitized Il4raF709 and Il4raF709 FcγRIIb −/− mice on the C57BL/6 background. ns, not significantly different. B) Serum levels IL-4. C) Intestinal mast cells. D) Specific IgE levels. E) Intestinal Treg frequencies. F) Intestinal Th2 frequencies. n=4–5 WT Control IgG, n=3–4 WT αOVA IgG, n=4–5 FcγRIIb −/− Control IgG and n=4–5 FcγRIIb −/− αOVA IgG. Data shown are from one of two independent experiments.

    Techniques Used: Mouse Assay

    Requirement for FcγRIIb in IgG-mediated suppression of IgE:FcεRI-triggered IL-4 expression. A) Supernatant levels of IL-4 in WT and FcγRIIb-deficient murine mast cells (C57BL/6 background, n=3) sensitized with polyclonal IgE αOVA and stimulated with OVA in the presence of monoclonal IgG2a αOVA. B) IL-13 secretion. C) mRNA levels of IL4 in human mast cells (n=3) sensitized with IgE αPN, and stimulated with PN in the presence of IgG αPN and/or αFcγRII (CD32) blocking antibody. D) IL-4 secretion by human mast cells. Data are representative of three independent experiments.
    Figure Legend Snippet: Requirement for FcγRIIb in IgG-mediated suppression of IgE:FcεRI-triggered IL-4 expression. A) Supernatant levels of IL-4 in WT and FcγRIIb-deficient murine mast cells (C57BL/6 background, n=3) sensitized with polyclonal IgE αOVA and stimulated with OVA in the presence of monoclonal IgG2a αOVA. B) IL-13 secretion. C) mRNA levels of IL4 in human mast cells (n=3) sensitized with IgE αPN, and stimulated with PN in the presence of IgG αPN and/or αFcγRII (CD32) blocking antibody. D) IL-4 secretion by human mast cells. Data are representative of three independent experiments.

    Techniques Used: Expressing, Blocking Assay

    Effect of IgG on food allergen sensitization. A) Anaphylaxis: Core body temperature decrease following enteral OVA challenge of sensitized BALB/c background Il4raF709 mice. n=4 saline, n=5 for OVA and OVA+IgG. B) Serum OVA-specific IgE. n.d., not detected. C) Levels of the mast cell granule protease, MMCP-1, in challenged mice. D) Serum IL-4 after challenge. E) Intestinal mast cell numbers. F) Small intestinal Th2 and G) Treg frequencies. Data shown are from one of three independent experiments.
    Figure Legend Snippet: Effect of IgG on food allergen sensitization. A) Anaphylaxis: Core body temperature decrease following enteral OVA challenge of sensitized BALB/c background Il4raF709 mice. n=4 saline, n=5 for OVA and OVA+IgG. B) Serum OVA-specific IgE. n.d., not detected. C) Levels of the mast cell granule protease, MMCP-1, in challenged mice. D) Serum IL-4 after challenge. E) Intestinal mast cell numbers. F) Small intestinal Th2 and G) Treg frequencies. Data shown are from one of three independent experiments.

    Techniques Used: Mouse Assay

    13) Product Images from "Multiple Components Rapidly Screened from Perilla Leaves Attenuate Asthma Airway Inflammation by Synergistic Targeting on Syk"

    Article Title: Multiple Components Rapidly Screened from Perilla Leaves Attenuate Asthma Airway Inflammation by Synergistic Targeting on Syk

    Journal: Journal of Inflammation Research

    doi: 10.2147/JIR.S281393

    Effects of Syk affinitive compounds from PLE on allergic airway inflammation in vivo. ( A ) Pathological changes in lung tissue were determined by H E staining (magnified ×100). ( B ) The scoring of inflammatory cells infiltration in H E stained lung tissues (n =20 was carried out using independent slices from 4 mice in each group). ( C ) Total and differential leukocyte counts in BALF (n = 8). ( D – H ) The levels of IL-6, TNF-α, IL-4, IFN-γ and the ratio of IL-4/IFN-γ in BALF (n = 8). ( I – L ) The levels of IgE, IgG1, IgG2a and IgG2b in serum (n = 8). Data were shown as mean ± SEM; ## P
    Figure Legend Snippet: Effects of Syk affinitive compounds from PLE on allergic airway inflammation in vivo. ( A ) Pathological changes in lung tissue were determined by H E staining (magnified ×100). ( B ) The scoring of inflammatory cells infiltration in H E stained lung tissues (n =20 was carried out using independent slices from 4 mice in each group). ( C ) Total and differential leukocyte counts in BALF (n = 8). ( D – H ) The levels of IL-6, TNF-α, IL-4, IFN-γ and the ratio of IL-4/IFN-γ in BALF (n = 8). ( I – L ) The levels of IgE, IgG1, IgG2a and IgG2b in serum (n = 8). Data were shown as mean ± SEM; ## P

    Techniques Used: In Vivo, Staining, Mouse Assay

    14) Product Images from "mTOR modulates the antibody response to provide cross-protective immunity to lethal influenza infections"

    Article Title: mTOR modulates the antibody response to provide cross-protective immunity to lethal influenza infections

    Journal: Nature immunology

    doi: 10.1038/ni.2741

    mTORC1 is required for Aicda transcription and B cell class-switching. (a–g) IgM + B cells were enriched from the spleen, CFSE labeled, and left unstimulated (US) or stimulated in vitro for 4 days with ( a–d ) LPS or ( e–g ) LPS and IL-4 in the presence of increasing doses of rapamycin. ( a ) Shown is the average number ± s.d. and ( b ) the percent ± s.d. of live divided B cells. The proportion of live B cells that are positive for ( c ) IgG2b, ( d ) IgG3, ( e ) IgG1 ( f ) IgE, and ( g ) IgM is shown. These data are representative of 5 separate experiments. ( h–j ) Control Rosa26 ERcreT and Raptor B − / − mice received injections of tamoxifen, i.p. daily for 4 days. Eight days after the last injection, IgM + B cells were harvested from spleens, stimulated for 24 hours with LPS and IL-4, with or without 0.5 ng/ml of rapamycin. ( h ) CFSE profile of control cells and ( i ) cell counts 24 hours after stimulation. ( j ) RNA was extracted and analyzed by qPCR for the level of Aicda transcripts relative to Cd79b transcripts. Data is a combination of 6 technical replicates and is representative of three biological replicates. (** P = 0.01, Mann-Whitney U test).
    Figure Legend Snippet: mTORC1 is required for Aicda transcription and B cell class-switching. (a–g) IgM + B cells were enriched from the spleen, CFSE labeled, and left unstimulated (US) or stimulated in vitro for 4 days with ( a–d ) LPS or ( e–g ) LPS and IL-4 in the presence of increasing doses of rapamycin. ( a ) Shown is the average number ± s.d. and ( b ) the percent ± s.d. of live divided B cells. The proportion of live B cells that are positive for ( c ) IgG2b, ( d ) IgG3, ( e ) IgG1 ( f ) IgE, and ( g ) IgM is shown. These data are representative of 5 separate experiments. ( h–j ) Control Rosa26 ERcreT and Raptor B − / − mice received injections of tamoxifen, i.p. daily for 4 days. Eight days after the last injection, IgM + B cells were harvested from spleens, stimulated for 24 hours with LPS and IL-4, with or without 0.5 ng/ml of rapamycin. ( h ) CFSE profile of control cells and ( i ) cell counts 24 hours after stimulation. ( j ) RNA was extracted and analyzed by qPCR for the level of Aicda transcripts relative to Cd79b transcripts. Data is a combination of 6 technical replicates and is representative of three biological replicates. (** P = 0.01, Mann-Whitney U test).

    Techniques Used: Labeling, In Vitro, Mouse Assay, Injection, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    Rapamycin modifies the antibody repertoire. Antigen microarrays were spotted with overlapping peptides that span the HA protein of both HKx31 and ΔVn1203. These were used to probe the IgM and IgG repertoire simultaneously using sera from animals sampled at 20 days following primary infection. Responses of 10 mice from each group were analyzed. ( a–b ) Samples were clustered using complete-linkage and the similarity between response patterns was defined as one minus the Spearman correlation coefficient between these vectors. Clustering dendrograms of response patterns of ( a ) IgM and ( b ) IgG to ΔVn1203 are depicted. ( c–f ) Specific antigens in which the responses between the rapamycin and PBS-treated mice had a p
    Figure Legend Snippet: Rapamycin modifies the antibody repertoire. Antigen microarrays were spotted with overlapping peptides that span the HA protein of both HKx31 and ΔVn1203. These were used to probe the IgM and IgG repertoire simultaneously using sera from animals sampled at 20 days following primary infection. Responses of 10 mice from each group were analyzed. ( a–b ) Samples were clustered using complete-linkage and the similarity between response patterns was defined as one minus the Spearman correlation coefficient between these vectors. Clustering dendrograms of response patterns of ( a ) IgM and ( b ) IgG to ΔVn1203 are depicted. ( c–f ) Specific antigens in which the responses between the rapamycin and PBS-treated mice had a p

    Techniques Used: Infection, Mouse Assay

    15) Product Images from "α-Galactosylceramide treatment before allergen sensitization promotes iNKT cell–mediated induction of Treg cells, preventing Th2 cell responses in murine asthma"

    Article Title: α-Galactosylceramide treatment before allergen sensitization promotes iNKT cell–mediated induction of Treg cells, preventing Th2 cell responses in murine asthma

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.005418

    α-GalCer treatment before OVA sensitization alleviates Th2 cell responses in an OVA-induced murine asthma model. WT mice, intraperitoneally treated with α-GalCer or PBS before OVA sensitization, were sacrificed 24 h after the final OVA challenge. A, histopathologic analysis of lung tissue H E and PAS staining. B, lung goblet cell hyperplasia is indicated as the number of PAS-positive cells per unit of length (mm) of the basement membrane. C, total and differential cell counting in BALF. Tot, total cell counts; Eos , eosinophils; Mac , macrophages; Neu , neutrophils; and Lym , lymphocytes. D, concentrations of IL-4, IL-5, IL-10, IL-13, and IFN-γ in BALFs were analyzed by ELISA. E, OVA-specific IgE and IgG1 concentrations in the serum. Data are expressed as the means ± S.D. of three independent experiments ( n = 5), and one representative experiment is indicated. *, p
    Figure Legend Snippet: α-GalCer treatment before OVA sensitization alleviates Th2 cell responses in an OVA-induced murine asthma model. WT mice, intraperitoneally treated with α-GalCer or PBS before OVA sensitization, were sacrificed 24 h after the final OVA challenge. A, histopathologic analysis of lung tissue H E and PAS staining. B, lung goblet cell hyperplasia is indicated as the number of PAS-positive cells per unit of length (mm) of the basement membrane. C, total and differential cell counting in BALF. Tot, total cell counts; Eos , eosinophils; Mac , macrophages; Neu , neutrophils; and Lym , lymphocytes. D, concentrations of IL-4, IL-5, IL-10, IL-13, and IFN-γ in BALFs were analyzed by ELISA. E, OVA-specific IgE and IgG1 concentrations in the serum. Data are expressed as the means ± S.D. of three independent experiments ( n = 5), and one representative experiment is indicated. *, p

    Techniques Used: Mouse Assay, Staining, Cell Counting, Enzyme-linked Immunosorbent Assay

    α-GalCer–mediated inhibition of Th2 cell responses requires iNKT cells in asthmatic mice. WT mice and CD1d −/− mice, immunized and challenged with OVA, were intraperitoneally injected with α-GalCer or PBS 24 h before the first sensitization. A, histopathological analysis of lung tissue H E and PAS staining. B, lung goblet cell hyperplasia is indicated as the number of PAS-positive cells per unit of length (mm) of the basement membrane. C, total and differential cell counting in BALF of mice. Tot , total cell counts; Eos , eosinophils; Mac , macrophages; Neu , neutrophils; and Lym , lymphocytes. D, concentrations of IL-4, IL-5, IL-10, IL-13, and IFN-γ in BALF from mice analyzed by ELISA. E, OVA-specific IgE and IgG1 concentrations in the serum. Data are expressed as the means ± S.D. of three independent experiments ( n = 5), and one representative experiment is indicated. *, p
    Figure Legend Snippet: α-GalCer–mediated inhibition of Th2 cell responses requires iNKT cells in asthmatic mice. WT mice and CD1d −/− mice, immunized and challenged with OVA, were intraperitoneally injected with α-GalCer or PBS 24 h before the first sensitization. A, histopathological analysis of lung tissue H E and PAS staining. B, lung goblet cell hyperplasia is indicated as the number of PAS-positive cells per unit of length (mm) of the basement membrane. C, total and differential cell counting in BALF of mice. Tot , total cell counts; Eos , eosinophils; Mac , macrophages; Neu , neutrophils; and Lym , lymphocytes. D, concentrations of IL-4, IL-5, IL-10, IL-13, and IFN-γ in BALF from mice analyzed by ELISA. E, OVA-specific IgE and IgG1 concentrations in the serum. Data are expressed as the means ± S.D. of three independent experiments ( n = 5), and one representative experiment is indicated. *, p

    Techniques Used: Inhibition, Mouse Assay, Injection, Staining, Cell Counting, Enzyme-linked Immunosorbent Assay

    α-GalCer treatment before HDM sensitization suppresses Th2 cell responses in HDM-induced murine asthma model. WT mice, intraperitoneally treated with α-GalCer or PBS before HDM sensitization, were sacrificed 3 days after the final HDM challenge. A, histopathological analysis of lung tissue H E and PAS staining. B, lung goblet cell hyperplasia is indicated as the number of PAS-positive cells per unit of length (mm) of the basement membrane. C, total and differential cell counting in BALF of mice. Tot, total cell counts; Eos, eosinophils; Mac, macrophages; Neu, neutrophils; and Lym, lymphocytes. D, BALF was collected 24 h after the final challenge, and the concentrations of IL-4, IL-5, IL-10, IL-13, and IFN-γ were analyzed by ELISA. E, HDM-specific IgE and IgG1 concentrations in the serum. Data are expressed as the means ± S.D. of three independent experiments ( n = 5), and one representative experiment is indicated. *, p
    Figure Legend Snippet: α-GalCer treatment before HDM sensitization suppresses Th2 cell responses in HDM-induced murine asthma model. WT mice, intraperitoneally treated with α-GalCer or PBS before HDM sensitization, were sacrificed 3 days after the final HDM challenge. A, histopathological analysis of lung tissue H E and PAS staining. B, lung goblet cell hyperplasia is indicated as the number of PAS-positive cells per unit of length (mm) of the basement membrane. C, total and differential cell counting in BALF of mice. Tot, total cell counts; Eos, eosinophils; Mac, macrophages; Neu, neutrophils; and Lym, lymphocytes. D, BALF was collected 24 h after the final challenge, and the concentrations of IL-4, IL-5, IL-10, IL-13, and IFN-γ were analyzed by ELISA. E, HDM-specific IgE and IgG1 concentrations in the serum. Data are expressed as the means ± S.D. of three independent experiments ( n = 5), and one representative experiment is indicated. *, p

    Techniques Used: Mouse Assay, Staining, Cell Counting, Enzyme-linked Immunosorbent Assay

    Treg cells can contribute to the inhibitory effects of α-GalCer treatment on Th2 cell responses in an OVA-induced murine asthma model. WT mice, intraperitoneally treated with α-GalCer before OVA sensitization, were delivered with anti-CD25 mAb or IgG isotype mAb 24 h before α-GalCer treatment and 48 h before OVA immunization. A, histopathological analysis of lung tissue H E and PAS staining. B, lung goblet cell hyperplasia is indicated as the number of PAS-positive cells per unit of length (mm) of the basement membrane. C, total and differential cell counting in BALF of mice. Tot, total cell counts; Eos, eosinophils; Mac, macrophages; Neu , neutrophils; and Lym, lymphocytes. D, concentrations of IL-4, IL-5, IL-10, IL-13, and IFN-γ in BALF from mice analyzed by ELISA. E, OVA-specific IgE and IgG1 concentrations in the serum. F, airway response to increasing concentrations of methacholine was examined. Data are expressed as the means ± S.D. of three independent experiments ( n = 5), and one representative experiment is indicated. *, p
    Figure Legend Snippet: Treg cells can contribute to the inhibitory effects of α-GalCer treatment on Th2 cell responses in an OVA-induced murine asthma model. WT mice, intraperitoneally treated with α-GalCer before OVA sensitization, were delivered with anti-CD25 mAb or IgG isotype mAb 24 h before α-GalCer treatment and 48 h before OVA immunization. A, histopathological analysis of lung tissue H E and PAS staining. B, lung goblet cell hyperplasia is indicated as the number of PAS-positive cells per unit of length (mm) of the basement membrane. C, total and differential cell counting in BALF of mice. Tot, total cell counts; Eos, eosinophils; Mac, macrophages; Neu , neutrophils; and Lym, lymphocytes. D, concentrations of IL-4, IL-5, IL-10, IL-13, and IFN-γ in BALF from mice analyzed by ELISA. E, OVA-specific IgE and IgG1 concentrations in the serum. F, airway response to increasing concentrations of methacholine was examined. Data are expressed as the means ± S.D. of three independent experiments ( n = 5), and one representative experiment is indicated. *, p

    Techniques Used: Mouse Assay, Staining, Cell Counting, Enzyme-linked Immunosorbent Assay

    Treg cells contribute to the inhibitory effects of α-GalCer on immunogenic maturation of LDCs in asthmatic mice. WT mice, intraperitoneally treated with α-GalCer before OVA sensitization, were delivered with anti-CD25 mAb or IgG isotype mAb 24 h before α-GalCer treatment and 48 h before OVA immunization. Then LDCs (CD11c + F4/80 − ) were isolated using a combination of magnetic microbead selection and flow cytometry. LDCs were quantified by CD11c and F4/80 staining using flow cytometry. A, expression of MHC II, CD80, CD86, and CD40 was assessed in CD11c + F4/80 − cells and isotype controls. B, expression levels of MHC II, CD80, CD86, and CD40 in CD11c + F4/80 − cells. C LDCs from the two groups of mice were cultured in vitro for 72 h. The concentrations of IL-12p70, IL-6, IL-10, and TNF-α in the culture supernatants were determined by ELISA. Data are represented as means ± S.D. from three independent experiments ( n = 5), and one representative experiment is indicated. *, p
    Figure Legend Snippet: Treg cells contribute to the inhibitory effects of α-GalCer on immunogenic maturation of LDCs in asthmatic mice. WT mice, intraperitoneally treated with α-GalCer before OVA sensitization, were delivered with anti-CD25 mAb or IgG isotype mAb 24 h before α-GalCer treatment and 48 h before OVA immunization. Then LDCs (CD11c + F4/80 − ) were isolated using a combination of magnetic microbead selection and flow cytometry. LDCs were quantified by CD11c and F4/80 staining using flow cytometry. A, expression of MHC II, CD80, CD86, and CD40 was assessed in CD11c + F4/80 − cells and isotype controls. B, expression levels of MHC II, CD80, CD86, and CD40 in CD11c + F4/80 − cells. C LDCs from the two groups of mice were cultured in vitro for 72 h. The concentrations of IL-12p70, IL-6, IL-10, and TNF-α in the culture supernatants were determined by ELISA. Data are represented as means ± S.D. from three independent experiments ( n = 5), and one representative experiment is indicated. *, p

    Techniques Used: Mouse Assay, Isolation, Selection, Flow Cytometry, Staining, Expressing, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay

    Treg cells induced by α-GalCer–activated iNKT cells from OVA-induced murine asthma may involve IL-2 in vitro . Lung iNKT cells (PBS-57/mCD1d + TCR-β + iNKT cells) were isolated from OVA-induced asthmatic mice intraperitoneally treated with α-GalCer before the first OVA sensitization, and spleen CD4 + CD25 − T cells were isolated from WT mice using magnetic microbead selection. iNKT cells were cultured alone or co-cultured with CD4 + CD25 − T cells in the presence of anti-IL-2 mAb or IgG isotype mAb for 72 h. A, flow cytometry determined the purity of lung iNKT cells, which were stained with both PBS-57/mCD1d and a mAb against TCR-β ( i.e. the proportion of iNKT cells was ∼96%). B, Treg cells of cellular components from culture medium were measured by CD4 and FoxP3 staining using flow cytometry. The gating used for Treg cells (CD4 + FoxP3 + Treg cells) and the corresponding percentages are shown in each dot plot. C, percentages of Treg cells from cellular components from culture medium. D, expression level of Foxp3 mRNA of cellular components from culture medium was analyzed by quantitative RT-PCR. E, concentration of IL-10 in culture supernatants was determined by ELISA. Data are shown as means ± S.D. of three independent experiments ( n = 15), and one representative experiment is indicated. *, p
    Figure Legend Snippet: Treg cells induced by α-GalCer–activated iNKT cells from OVA-induced murine asthma may involve IL-2 in vitro . Lung iNKT cells (PBS-57/mCD1d + TCR-β + iNKT cells) were isolated from OVA-induced asthmatic mice intraperitoneally treated with α-GalCer before the first OVA sensitization, and spleen CD4 + CD25 − T cells were isolated from WT mice using magnetic microbead selection. iNKT cells were cultured alone or co-cultured with CD4 + CD25 − T cells in the presence of anti-IL-2 mAb or IgG isotype mAb for 72 h. A, flow cytometry determined the purity of lung iNKT cells, which were stained with both PBS-57/mCD1d and a mAb against TCR-β ( i.e. the proportion of iNKT cells was ∼96%). B, Treg cells of cellular components from culture medium were measured by CD4 and FoxP3 staining using flow cytometry. The gating used for Treg cells (CD4 + FoxP3 + Treg cells) and the corresponding percentages are shown in each dot plot. C, percentages of Treg cells from cellular components from culture medium. D, expression level of Foxp3 mRNA of cellular components from culture medium was analyzed by quantitative RT-PCR. E, concentration of IL-10 in culture supernatants was determined by ELISA. Data are shown as means ± S.D. of three independent experiments ( n = 15), and one representative experiment is indicated. *, p

    Techniques Used: In Vitro, Isolation, Mouse Assay, Selection, Cell Culture, Flow Cytometry, Staining, Expressing, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

    16) Product Images from "IgA and IgG1 Specific to Vi Polysaccharide of Salmonella Typhi Correlate With Protection Status in a Typhoid Fever Controlled Human Infection Model"

    Article Title: IgA and IgG1 Specific to Vi Polysaccharide of Salmonella Typhi Correlate With Protection Status in a Typhoid Fever Controlled Human Infection Model

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02582

    Vi polysaccharide IgG1 avidity higher in protected individuals. ViBIOT IgG1 avidity index at pH 3.0 in positive vaccine responders by vaccine group over time (A) and by diagnosed/protected outcome at day of challenge (B) . Avidity index in positive vaccine responses of anti-ViBIOT IgG1 over time (C) from 4 weeks post-vaccination (D0) to 6 months post-challenge (D180) by vaccine group and protection status. Bolded lines indicate median avidity index and faint lines indicate individual level data. Data points are representative of n = 2 independent experiments (each with n = 2 technical replicates). nt indicates not tested for statistical significance.
    Figure Legend Snippet: Vi polysaccharide IgG1 avidity higher in protected individuals. ViBIOT IgG1 avidity index at pH 3.0 in positive vaccine responders by vaccine group over time (A) and by diagnosed/protected outcome at day of challenge (B) . Avidity index in positive vaccine responses of anti-ViBIOT IgG1 over time (C) from 4 weeks post-vaccination (D0) to 6 months post-challenge (D180) by vaccine group and protection status. Bolded lines indicate median avidity index and faint lines indicate individual level data. Data points are representative of n = 2 independent experiments (each with n = 2 technical replicates). nt indicates not tested for statistical significance.

    Techniques Used:

    Different immune signatures of protection status for Vi-PS and Vi-TT. Variables in analysis of immune signatures between vaccine groups include magnitude, fold-change, and avidity of IgA1, IgA2, IgG1, IgG2, and IgG3 to Vi-Biot and nViPS. Volcano plot of FDR-corrected p -value and fold difference by diagnosed/protected outcome for Vi-PS vaccinees (A) and Vi-TT vaccinees (B) . Red dots represent FDR-corrected p -values
    Figure Legend Snippet: Different immune signatures of protection status for Vi-PS and Vi-TT. Variables in analysis of immune signatures between vaccine groups include magnitude, fold-change, and avidity of IgA1, IgA2, IgG1, IgG2, and IgG3 to Vi-Biot and nViPS. Volcano plot of FDR-corrected p -value and fold difference by diagnosed/protected outcome for Vi-PS vaccinees (A) and Vi-TT vaccinees (B) . Red dots represent FDR-corrected p -values

    Techniques Used:

    IgA dominates the vaccine-elicited antibody response to Vi polysaccharide. Concentration of antigen-specific, vaccine-induced IgA, IgG1, IgG2, and IgG3 to ViBIOT (A) and tetanus toxoid (B) by vaccine group, Vi-PS in black and Vi-TT in green, of positive vaccine responders only. Percent positive responders indicated post-vaccination at D0. Vi-PS vaccinees exhibited no vaccine-induced tetanus toxoid response. Data points are representative of n = 2 independent experiments (each with n = 2 technical replicates). Fold-change in magnitude of the response to Vi from Baseline to Day of Challenge across subclasses by vaccine group (C) . A principal components analysis with all tetanus and Vi responses included (D) with a scatter plot of the first (PC1) and second (PC2) principal components is shown. Each measurement from a Vi-PS ( n = 35 participants) or a Vi-TT ( n = 37 participants) vaccinee is represented by a black or green dot, respectively. Ellipses represent 95% confidence regions.
    Figure Legend Snippet: IgA dominates the vaccine-elicited antibody response to Vi polysaccharide. Concentration of antigen-specific, vaccine-induced IgA, IgG1, IgG2, and IgG3 to ViBIOT (A) and tetanus toxoid (B) by vaccine group, Vi-PS in black and Vi-TT in green, of positive vaccine responders only. Percent positive responders indicated post-vaccination at D0. Vi-PS vaccinees exhibited no vaccine-induced tetanus toxoid response. Data points are representative of n = 2 independent experiments (each with n = 2 technical replicates). Fold-change in magnitude of the response to Vi from Baseline to Day of Challenge across subclasses by vaccine group (C) . A principal components analysis with all tetanus and Vi responses included (D) with a scatter plot of the first (PC1) and second (PC2) principal components is shown. Each measurement from a Vi-PS ( n = 35 participants) or a Vi-TT ( n = 37 participants) vaccinee is represented by a black or green dot, respectively. Ellipses represent 95% confidence regions.

    Techniques Used: Concentration Assay

    17) Product Images from "Asthma-Associated Long TSLP Inhibits the Production of IgA"

    Article Title: Asthma-Associated Long TSLP Inhibits the Production of IgA

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22073592

    The role of retinoic acid (RA) in inducing secretion of IgA. CD19 B cells were cultured for 7 days and stained for CD20 and CD38 ( A ) in the presence or absence of RA and TSLP ( B ) for 3 donors in a single experiment. B cells were cultured for 11 days and supernatants analyzed for concentrations of IgA ( C ) and IgG1 ( D ) for seven donors in two independent experiments. Graphs show mean and standard error of the mean, and statistical test results are obtained from ANOVA with Sidaks correction for multiple testing.
    Figure Legend Snippet: The role of retinoic acid (RA) in inducing secretion of IgA. CD19 B cells were cultured for 7 days and stained for CD20 and CD38 ( A ) in the presence or absence of RA and TSLP ( B ) for 3 donors in a single experiment. B cells were cultured for 11 days and supernatants analyzed for concentrations of IgA ( C ) and IgG1 ( D ) for seven donors in two independent experiments. Graphs show mean and standard error of the mean, and statistical test results are obtained from ANOVA with Sidaks correction for multiple testing.

    Techniques Used: Cell Culture, Staining

    Flow cytometric analysis of B cells after 7 days of stimulation. An example of IgG and IgA flow cytometric analysis ( A ) and summarized data showing mean and standard error of the mean ( B ) of 4–5 donors are shown from two independent experiments. The overlaying bar indicates that the differences of the five small bars are all p
    Figure Legend Snippet: Flow cytometric analysis of B cells after 7 days of stimulation. An example of IgG and IgA flow cytometric analysis ( A ) and summarized data showing mean and standard error of the mean ( B ) of 4–5 donors are shown from two independent experiments. The overlaying bar indicates that the differences of the five small bars are all p

    Techniques Used:

    IgG and IgA production by naïve and memory B cells stimulated with loTSLP or shTSLP. Naïve and memory B cells were stimulated for 11 days using the T cell dependent protocol and supernatants analyzed for concentrations of IgG1 ( A ) and IgA ( B ). Boxplots show mean, second, and third (box) and first and fourth percentile of seven donors tested in two independent experiments.
    Figure Legend Snippet: IgG and IgA production by naïve and memory B cells stimulated with loTSLP or shTSLP. Naïve and memory B cells were stimulated for 11 days using the T cell dependent protocol and supernatants analyzed for concentrations of IgG1 ( A ) and IgA ( B ). Boxplots show mean, second, and third (box) and first and fourth percentile of seven donors tested in two independent experiments.

    Techniques Used:

    Regulation of Ig secretion by IL-4, IL-13, and TSLP. The concentrations of IgM ( A ), IgA ( B ), IgG1 ( C ), IgG2 ( D ), IgG3 ( E ), and IgG4 ( F ) in B cell culture supernatants after 11 days of stimulation using the T cell-dependent protocol using irradiated CD40L expressing cells. Mean concentrations and standard error of the mean are shown. Statistical test results are from ANOVA with Sidaks correction for multiple testing using log-transformed concentration data of four to six donors and two independent experiments.
    Figure Legend Snippet: Regulation of Ig secretion by IL-4, IL-13, and TSLP. The concentrations of IgM ( A ), IgA ( B ), IgG1 ( C ), IgG2 ( D ), IgG3 ( E ), and IgG4 ( F ) in B cell culture supernatants after 11 days of stimulation using the T cell-dependent protocol using irradiated CD40L expressing cells. Mean concentrations and standard error of the mean are shown. Statistical test results are from ANOVA with Sidaks correction for multiple testing using log-transformed concentration data of four to six donors and two independent experiments.

    Techniques Used: Cell Culture, Irradiation, Expressing, Transformation Assay, Concentration Assay

    18) Product Images from "Altered marginal zone and innate-like B cells in aged senescence-accelerated SAMP8 mice with defective IgG1 responses"

    Article Title: Altered marginal zone and innate-like B cells in aged senescence-accelerated SAMP8 mice with defective IgG1 responses

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.351

    Immunoglobulin isotypes in serum from SAMR1 and SAMP8 mice determined by ELISA. ( a ) Serum IgM and IgG1 from 2- and 10-month-old SAMR1 and SAMP8 mice. ( b ) IgG2a, IgG2b and IgG3 isotypes were quantified on sera from 10-month-old SAMP8 and SAMR1 mice. Data are measurements of individual mice. Means are indicated by the horizontal lines. Comparisons were made with the unpaired two-tailed Student’s t -test: ** P
    Figure Legend Snippet: Immunoglobulin isotypes in serum from SAMR1 and SAMP8 mice determined by ELISA. ( a ) Serum IgM and IgG1 from 2- and 10-month-old SAMR1 and SAMP8 mice. ( b ) IgG2a, IgG2b and IgG3 isotypes were quantified on sera from 10-month-old SAMP8 and SAMR1 mice. Data are measurements of individual mice. Means are indicated by the horizontal lines. Comparisons were made with the unpaired two-tailed Student’s t -test: ** P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Plasma cell B cell maturation and distribution of the memory B-cell compartments in aged SAMP8 mice. ( a ) Cells were stained with anti-CD19-Violet-421, anti-CD45R-PE and anti-CD138-APC. Absolute numbers of CD138 + plasmablasts were calculated from flow cytometry as in Figure 1c . Data are mean±S.E.M., ( n =4). ( b ) IgG1-specific ELISPOT analysis (see ‘Materials and Methods’) of purified samples of B1REL and B2 cells obtained from aged SAMR1 and SAMP8 mice. The numbers of IgG1- antibody secreting cells (ASCs) are shown. Data are mean±S.E.M., ( n =4). ( c ) RT-qPCR analyses were performed for Pax5, Xbp-1, Blimp1 and AID transcript expression in purified B1REL cells from 10-month-old SAMR1 and SAMP8 mice (see ‘Materials and Methods’). The Bio-Rad CFX Manager software (Bio-Rad, Hercules, CA, USA) was used to calculate the C T of each reaction. The amount of specific transcripts in each cDNA sample was determined as the 2 −ΔΔ C T , relative to that of the HPRT transcripts and normalized to those obtained for 2-month-old BALB/c samples used as reference. Data are mean±S.E.M. ( n =6–10 performed in duplicates). ( d–f ) Flow cytometry analyses 10-month-old SAMR1 and SAMP8 mice spleen samples. Representative dot-plots are shown for each mouse. Numbers inside the plots are frequencies of each population (mean±S.E.M.: n =4 for T FH cells and for GL7 + and IgD+ cells, and n =3 for memBC). Fluorescence scales are logarithmic. ( d ) Staining was performed with anti-CD4-APC, anti-CXCR5-Violet-421 and anti-PD1-biotynilated and revealed with streptavidin-PE-Cy7. T FH cells were gated as CD4 + CXCR5 + PD1 + cells (boxes). ( e ) MemBC were determined as CD11b − Gr1 − CD138 − IgM − IgD − CD19 + CD38 + IgG1 + by using anti-CD11b- and anti-Gr1-PE-Cy7, anti-CD138-PE, anti-IgM- and anti-IgD-FITC, anti-CD19-Violet-421, anti-CD38-APC and rat anti-mouse IgG1-biotynilated (clone A85-1, rat IgG1/k). The FMO control was a biotinylated rat IgG1/ k (clone R3-34). Biotynilated Abs were revealed with streptavidin-APC-Cy7. The histograms display the IgG1 + cells among the CD138 − CD38 + cells (empty, FMO isotype control; filled gray, IgG1 + cells). ( f ) Staining was performed with anti-CD19-Violet-421, anti-GL7-PE and anti-IgD-FITC, to determine GL7 + cells and naive IgD + cells on gated CD19 cells. ( g ) Bar graph shows the absolute number/spleen of T FH , GL7 + , memBC and naive B cells (right Y scale). These absolute numbers were calculated from the frequencies of each population. Shown are the mean±S.E.M. ( n =4 and 3 for memBC). The group comparisons were made using the unpaired two-tailed Student’s t -test: * P
    Figure Legend Snippet: Plasma cell B cell maturation and distribution of the memory B-cell compartments in aged SAMP8 mice. ( a ) Cells were stained with anti-CD19-Violet-421, anti-CD45R-PE and anti-CD138-APC. Absolute numbers of CD138 + plasmablasts were calculated from flow cytometry as in Figure 1c . Data are mean±S.E.M., ( n =4). ( b ) IgG1-specific ELISPOT analysis (see ‘Materials and Methods’) of purified samples of B1REL and B2 cells obtained from aged SAMR1 and SAMP8 mice. The numbers of IgG1- antibody secreting cells (ASCs) are shown. Data are mean±S.E.M., ( n =4). ( c ) RT-qPCR analyses were performed for Pax5, Xbp-1, Blimp1 and AID transcript expression in purified B1REL cells from 10-month-old SAMR1 and SAMP8 mice (see ‘Materials and Methods’). The Bio-Rad CFX Manager software (Bio-Rad, Hercules, CA, USA) was used to calculate the C T of each reaction. The amount of specific transcripts in each cDNA sample was determined as the 2 −ΔΔ C T , relative to that of the HPRT transcripts and normalized to those obtained for 2-month-old BALB/c samples used as reference. Data are mean±S.E.M. ( n =6–10 performed in duplicates). ( d–f ) Flow cytometry analyses 10-month-old SAMR1 and SAMP8 mice spleen samples. Representative dot-plots are shown for each mouse. Numbers inside the plots are frequencies of each population (mean±S.E.M.: n =4 for T FH cells and for GL7 + and IgD+ cells, and n =3 for memBC). Fluorescence scales are logarithmic. ( d ) Staining was performed with anti-CD4-APC, anti-CXCR5-Violet-421 and anti-PD1-biotynilated and revealed with streptavidin-PE-Cy7. T FH cells were gated as CD4 + CXCR5 + PD1 + cells (boxes). ( e ) MemBC were determined as CD11b − Gr1 − CD138 − IgM − IgD − CD19 + CD38 + IgG1 + by using anti-CD11b- and anti-Gr1-PE-Cy7, anti-CD138-PE, anti-IgM- and anti-IgD-FITC, anti-CD19-Violet-421, anti-CD38-APC and rat anti-mouse IgG1-biotynilated (clone A85-1, rat IgG1/k). The FMO control was a biotinylated rat IgG1/ k (clone R3-34). Biotynilated Abs were revealed with streptavidin-APC-Cy7. The histograms display the IgG1 + cells among the CD138 − CD38 + cells (empty, FMO isotype control; filled gray, IgG1 + cells). ( f ) Staining was performed with anti-CD19-Violet-421, anti-GL7-PE and anti-IgD-FITC, to determine GL7 + cells and naive IgD + cells on gated CD19 cells. ( g ) Bar graph shows the absolute number/spleen of T FH , GL7 + , memBC and naive B cells (right Y scale). These absolute numbers were calculated from the frequencies of each population. Shown are the mean±S.E.M. ( n =4 and 3 for memBC). The group comparisons were made using the unpaired two-tailed Student’s t -test: * P

    Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunospot, Purification, Quantitative RT-PCR, Expressing, Software, Fluorescence, Two Tailed Test

    In vitro and in vivo responses of B-cell subsets from SAMP8 mice stimulated with LPS ( a ) and ( b ). The indicated B-cell subsets identified as in Figure 1b from spleens of 10-month-old SAMP8 and SAMR1 mice were FACS-purified, and then labeled with the CellTrace Violet kit before culturing for 72 h in the presence or absence of LPS (see Materials and Methods). After culture, the cells were washed and stained to detect CD138 and the incorporated violet dye by flow cytometry. ( a ) Representative results from B1REL, ABC and B2 cells after LPS stimulation are shown. Fluorescence scales are logarithmic. The numbers in the plots are the frequency in each quadrant. Data are means±S.E.M. ( n =3). ( b ) The IgM and IgG1 secreted into the culture medium after 72 h were determined by ELISA. Data are means±S.E.M. ( n =3). Comparisons were made using a two-tailed Student’s t -test: * P
    Figure Legend Snippet: In vitro and in vivo responses of B-cell subsets from SAMP8 mice stimulated with LPS ( a ) and ( b ). The indicated B-cell subsets identified as in Figure 1b from spleens of 10-month-old SAMP8 and SAMR1 mice were FACS-purified, and then labeled with the CellTrace Violet kit before culturing for 72 h in the presence or absence of LPS (see Materials and Methods). After culture, the cells were washed and stained to detect CD138 and the incorporated violet dye by flow cytometry. ( a ) Representative results from B1REL, ABC and B2 cells after LPS stimulation are shown. Fluorescence scales are logarithmic. The numbers in the plots are the frequency in each quadrant. Data are means±S.E.M. ( n =3). ( b ) The IgM and IgG1 secreted into the culture medium after 72 h were determined by ELISA. Data are means±S.E.M. ( n =3). Comparisons were made using a two-tailed Student’s t -test: * P

    Techniques Used: In Vitro, In Vivo, Mouse Assay, FACS, Purification, Labeling, Staining, Flow Cytometry, Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Two Tailed Test

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    Article Title: SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization
    Article Snippet: The binding of recombinant antibodies to SARS-2-S was determined by flow cytometry with 293T cells stably transfected with plasmid pWHE469-SARS-CoV2 containing the ORF of the spike protein of SARS-CoV-2 isolate Wuhan-Hu-1 (position 21580 – 25400 from GenBank NC_045512) and a GFP reporter plasmid under the control of a doxycycline-inducible promotor ( ). .. Briefly, 293T cells were stained with the recombinant human IgG1 antibodies in FACS buffer (PBS with 0.5% bovine serum albumin and 1 nmol sodium azide) for 20 minutes in ice, washed, incubated with an Alexa Fluor 647-labeled mouse monoclonal antibody against the human IgG1-Fc (Biolegend, San Diego, USA, cat #409320) and analyzed in a Gallios flow cytometer (Beckman Coulter, Brea, California, USA respectively). .. Production of rhabdoviral pseudotype particles and transduction of target cellsRhabdoviral pseudotype particles bearing WT or mutant SARS-2-S, VSV-G or no viral protein (negative control) were prepared according to a published protocol ( ) and involved a replication-deficient VSV vector that lacks the genetic information for VSV-G and instead codes for two reporter proteins, enhanced green fluorescent protein and firefly luciferase (FLuc), VSV∗ΔG-FLuc (kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern, Switzerland) ( ).

    Article Title: SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization
    Article Snippet: The binding of recombinant antibodies to SARS-2-S was determined by flow cytometry with 293T cells stably transfected with plasmid pWHE469-SARS-CoV2 containing the ORF of the spike protein of SARS-CoV-2 isolate Wuhan-Hu-1 (position 21580 – 25400 from GenBank NC_045512) and a GFP reporter plasmid under the control of a doxycycline-inducible promotor ( ). .. Briefly, 293T cells were stained with the recombinant human IgG1 antibodies in FACS buffer (PBS with 0.5% bovine serum albumin and 1 nmol sodium azide) for 20 minutes in ice, washed, incubated with an Alexa Fluor 647-labeled mouse monoclonal antibody against the human IgG1-Fc (Biolegend, San Diego, USA, cat #409320) and analyzed in a Gallios flow cytometer (Beckman Coulter, Brea, California, USA respectively). .. Production of rhabdoviral pseudotype particles and transduction of target cells Rhabdoviral pseudotype particles bearing WT or mutant SARS-2-S, VSV-G or no viral protein (negative control) were prepared according to a published protocol ( ) based on a replication-deficient VSV vector that lacks the genetic information for VSV-G and instead codes for two reporter proteins, enhanced green fluorescent protein and firefly luciferase (FLuc), VSV∗ ΔG-FLuc (kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern, Switzerland) ( ).

    Staining:

    Article Title: SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization
    Article Snippet: The binding of recombinant antibodies to SARS-2-S was determined by flow cytometry with 293T cells stably transfected with plasmid pWHE469-SARS-CoV2 containing the ORF of the spike protein of SARS-CoV-2 isolate Wuhan-Hu-1 (position 21580 – 25400 from GenBank NC_045512) and a GFP reporter plasmid under the control of a doxycycline-inducible promotor ( ). .. Briefly, 293T cells were stained with the recombinant human IgG1 antibodies in FACS buffer (PBS with 0.5% bovine serum albumin and 1 nmol sodium azide) for 20 minutes in ice, washed, incubated with an Alexa Fluor 647-labeled mouse monoclonal antibody against the human IgG1-Fc (Biolegend, San Diego, USA, cat #409320) and analyzed in a Gallios flow cytometer (Beckman Coulter, Brea, California, USA respectively). .. Production of rhabdoviral pseudotype particles and transduction of target cellsRhabdoviral pseudotype particles bearing WT or mutant SARS-2-S, VSV-G or no viral protein (negative control) were prepared according to a published protocol ( ) and involved a replication-deficient VSV vector that lacks the genetic information for VSV-G and instead codes for two reporter proteins, enhanced green fluorescent protein and firefly luciferase (FLuc), VSV∗ΔG-FLuc (kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern, Switzerland) ( ).

    Article Title: SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization
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    FACS:

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    Article Title: SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization
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    Incubation:

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    Flow Cytometry:

    Article Title: SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization
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    BioLegend igg2b
    CC induce PTF1.2 dependent on the TF pathway. ( A ) PTF1.2 induction over time (60, 120, and 240 min) upon exposure to CC 2000 μg/ml, PBS/HSA, E. coli (1 × 10 7 particles/ml), or glass ( n = 7 donors) in human whole blood. ( B ) Effect of TF inhibition following exposure of CC (500, 1000, and 2000 μg/ml) or E.coli after addition of the functional-grade inhibitory Ab against TF (anti-TF) or the corresponding Ultra-LEAF purified IgG1κ control Ab (Ctr <t>IgG)</t> for 240 min in human whole blood ( n = 6 donors). ( C ) Effect of factor XII inhibition by CTI (40 μg/ml) in PFP following incubation with CC (2000 μg/ml) or glass for 60 min ( n = 5). Effect of CC on platelet aggregation in human whole blood measured by electrical impedance aggregometry in ( D ) by CC (2000 μg/ml) or its controls, HSA/PBS, or TRAP ( n = 6), ( E ) by additional stimulation with ADP ( n = 3), and in ( F ) with TRAP following 6 min of incubation ( n = 3), given as arbitrary units × min. In all experiments, thrombin was inhibited with lepirudin. All data are given as means ± SEM. * p
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    BioLegend igg1
    Humoral immune responses after vaccination in young and aged Sost -/- mice A-E) Analysis of T-independent B cell responses: Experimental design (A); Analysis of NP-specific IgM titers and IgG3 titers in young WT and Sost -/- mice (B, C), Analysis of NP-specific IgM titers and IgG3 titers in aged WT and Sost -/- mice (D, E). F-J) Analysis of T-dependent B cell responses: Experimental design (F); Analysis of NP-specific IgM titers and <t>IgG1</t> titers in young WT and Sost -/- mice (G, H), Analysis of NP-specific IgM titers and IgG1 titers in aged WT and Sost -/- mice (I, J). Asterisks indicate statistical significance: * p
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    92
    BioLegend rat igg1 biotin
    IgGs bind to thymic stromal components in non-obese diabetic (NOD) mice. (A,B) Representative confocal immunofluorescence microscopy images of thymi sections of NOD [ (A) , I–III] and B6 mice [ (B) , I–II] examined for cytokeratin V (red), murine <t>IgG</t> (green), and the DNA-intercalating dye DAPI (white). A total of six 11-week-old NOD mice and five 11-week-old B6 mice, two sections per mouse were examined. [ (A) , I–II] is derived from different NOD mice. The confocal fluorescent image in AI was obtained with a Plan-Apochromat 20× objective to give a broader view of the extent of immunoglobulin bound to thymic stroma, arrows indicating some of the cells co-positive for cytokeratin V and mouse IgG. The confocal fluorescent images in AII and AIII were obtained with a Plan-Apochromat 63× objective. For panel (B) , the confocal fluorescent image was obtained using a Plan-Apochromat 20× objective. (C) Quantification of murine Ig-bound to stromal cells of age-matched 11-week-old, female NOD or B6 mice. Confocal immunofluorescence microscopy images were subjected to StrataQuest V64 analysis, a total of 2–3 × 10 4 DAPI + cells/mm 2 were counted, and the mean fluorescence intensity of DAPI + cells versus mean fluorescence intensity of anti-Ig is presented as a scattergram. The data shown are representative of two independent mice examined giving similar results.
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    BioLegend il 7rα
    The number of ETPs and TECs was significantly increased in allo-BMT recipients after rIL-7/HGFβ treatment. Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice and treated with cytokines as in Figure 1 . On day 30 after BMT, ETPs and TECs were analyzed. (A) Representative flow cytometric profiles showing the percentage of donor-origin lineage - c-kit + <t>IL-7Rα</t> - CD44 + CD25 - ETPs in total thymocytes. (B) Number of donor-origin ETPs in the cytokine-treated BMT mice. (C) Representative flow cytometric profiles showing the percentage of CD45 - EpCAM + MHC II + Ly51 + cTECs and CD45 - EpCAM + MHC II + Ly51 - mTECs in total TECs of the rIL-7/HGFβ-treated BMT mice. (D) Number of total TECs, cTEC and mTECs in the cytokine-treated BMT mice. The data are representative of 2 independent experiments with 4-6 mice per group. * P
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    CC induce PTF1.2 dependent on the TF pathway. ( A ) PTF1.2 induction over time (60, 120, and 240 min) upon exposure to CC 2000 μg/ml, PBS/HSA, E. coli (1 × 10 7 particles/ml), or glass ( n = 7 donors) in human whole blood. ( B ) Effect of TF inhibition following exposure of CC (500, 1000, and 2000 μg/ml) or E.coli after addition of the functional-grade inhibitory Ab against TF (anti-TF) or the corresponding Ultra-LEAF purified IgG1κ control Ab (Ctr IgG) for 240 min in human whole blood ( n = 6 donors). ( C ) Effect of factor XII inhibition by CTI (40 μg/ml) in PFP following incubation with CC (2000 μg/ml) or glass for 60 min ( n = 5). Effect of CC on platelet aggregation in human whole blood measured by electrical impedance aggregometry in ( D ) by CC (2000 μg/ml) or its controls, HSA/PBS, or TRAP ( n = 6), ( E ) by additional stimulation with ADP ( n = 3), and in ( F ) with TRAP following 6 min of incubation ( n = 3), given as arbitrary units × min. In all experiments, thrombin was inhibited with lepirudin. All data are given as means ± SEM. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Cholesterol Crystals Induce Coagulation Activation through Complement-Dependent Expression of Monocytic Tissue Factor

    doi: 10.4049/jimmunol.1900503

    Figure Lengend Snippet: CC induce PTF1.2 dependent on the TF pathway. ( A ) PTF1.2 induction over time (60, 120, and 240 min) upon exposure to CC 2000 μg/ml, PBS/HSA, E. coli (1 × 10 7 particles/ml), or glass ( n = 7 donors) in human whole blood. ( B ) Effect of TF inhibition following exposure of CC (500, 1000, and 2000 μg/ml) or E.coli after addition of the functional-grade inhibitory Ab against TF (anti-TF) or the corresponding Ultra-LEAF purified IgG1κ control Ab (Ctr IgG) for 240 min in human whole blood ( n = 6 donors). ( C ) Effect of factor XII inhibition by CTI (40 μg/ml) in PFP following incubation with CC (2000 μg/ml) or glass for 60 min ( n = 5). Effect of CC on platelet aggregation in human whole blood measured by electrical impedance aggregometry in ( D ) by CC (2000 μg/ml) or its controls, HSA/PBS, or TRAP ( n = 6), ( E ) by additional stimulation with ADP ( n = 3), and in ( F ) with TRAP following 6 min of incubation ( n = 3), given as arbitrary units × min. In all experiments, thrombin was inhibited with lepirudin. All data are given as means ± SEM. * p

    Article Snippet: Isotype controls were purified mouse Igs IgG1 (clone MOPC-21), IgG2a (clone MOPC-172), and IgG2b (clone MPC-11) from BioLegend.

    Techniques: Inhibition, Functional Assay, Purification, Incubation

    Humoral immune responses after vaccination in young and aged Sost -/- mice A-E) Analysis of T-independent B cell responses: Experimental design (A); Analysis of NP-specific IgM titers and IgG3 titers in young WT and Sost -/- mice (B, C), Analysis of NP-specific IgM titers and IgG3 titers in aged WT and Sost -/- mice (D, E). F-J) Analysis of T-dependent B cell responses: Experimental design (F); Analysis of NP-specific IgM titers and IgG1 titers in young WT and Sost -/- mice (G, H), Analysis of NP-specific IgM titers and IgG1 titers in aged WT and Sost -/- mice (I, J). Asterisks indicate statistical significance: * p

    Journal: bioRxiv

    Article Title: Sclerostin Deficiency Alters Peripheral B Lymphocyte Responses in Mice

    doi: 10.1101/357772

    Figure Lengend Snippet: Humoral immune responses after vaccination in young and aged Sost -/- mice A-E) Analysis of T-independent B cell responses: Experimental design (A); Analysis of NP-specific IgM titers and IgG3 titers in young WT and Sost -/- mice (B, C), Analysis of NP-specific IgM titers and IgG3 titers in aged WT and Sost -/- mice (D, E). F-J) Analysis of T-dependent B cell responses: Experimental design (F); Analysis of NP-specific IgM titers and IgG1 titers in young WT and Sost -/- mice (G, H), Analysis of NP-specific IgM titers and IgG1 titers in aged WT and Sost -/- mice (I, J). Asterisks indicate statistical significance: * p

    Article Snippet: ELISA plates (Fisherbrand) were plated with serial dilutions of purified mouse IgM, IgG3 or IgG1 (BioLegend) to create a standard curve.

    Techniques: Mouse Assay

    IgGs bind to thymic stromal components in non-obese diabetic (NOD) mice. (A,B) Representative confocal immunofluorescence microscopy images of thymi sections of NOD [ (A) , I–III] and B6 mice [ (B) , I–II] examined for cytokeratin V (red), murine IgG (green), and the DNA-intercalating dye DAPI (white). A total of six 11-week-old NOD mice and five 11-week-old B6 mice, two sections per mouse were examined. [ (A) , I–II] is derived from different NOD mice. The confocal fluorescent image in AI was obtained with a Plan-Apochromat 20× objective to give a broader view of the extent of immunoglobulin bound to thymic stroma, arrows indicating some of the cells co-positive for cytokeratin V and mouse IgG. The confocal fluorescent images in AII and AIII were obtained with a Plan-Apochromat 63× objective. For panel (B) , the confocal fluorescent image was obtained using a Plan-Apochromat 20× objective. (C) Quantification of murine Ig-bound to stromal cells of age-matched 11-week-old, female NOD or B6 mice. Confocal immunofluorescence microscopy images were subjected to StrataQuest V64 analysis, a total of 2–3 × 10 4 DAPI + cells/mm 2 were counted, and the mean fluorescence intensity of DAPI + cells versus mean fluorescence intensity of anti-Ig is presented as a scattergram. The data shown are representative of two independent mice examined giving similar results.

    Journal: Frontiers in Immunology

    Article Title: Thymic B Cell-Mediated Attack of Thymic Stroma Precedes Type 1 Diabetes Development

    doi: 10.3389/fimmu.2018.01281

    Figure Lengend Snippet: IgGs bind to thymic stromal components in non-obese diabetic (NOD) mice. (A,B) Representative confocal immunofluorescence microscopy images of thymi sections of NOD [ (A) , I–III] and B6 mice [ (B) , I–II] examined for cytokeratin V (red), murine IgG (green), and the DNA-intercalating dye DAPI (white). A total of six 11-week-old NOD mice and five 11-week-old B6 mice, two sections per mouse were examined. [ (A) , I–II] is derived from different NOD mice. The confocal fluorescent image in AI was obtained with a Plan-Apochromat 20× objective to give a broader view of the extent of immunoglobulin bound to thymic stroma, arrows indicating some of the cells co-positive for cytokeratin V and mouse IgG. The confocal fluorescent images in AII and AIII were obtained with a Plan-Apochromat 63× objective. For panel (B) , the confocal fluorescent image was obtained using a Plan-Apochromat 20× objective. (C) Quantification of murine Ig-bound to stromal cells of age-matched 11-week-old, female NOD or B6 mice. Confocal immunofluorescence microscopy images were subjected to StrataQuest V64 analysis, a total of 2–3 × 10 4 DAPI + cells/mm 2 were counted, and the mean fluorescence intensity of DAPI + cells versus mean fluorescence intensity of anti-Ig is presented as a scattergram. The data shown are representative of two independent mice examined giving similar results.

    Article Snippet: The gates were defined using fluorescence minus one and isotype controls: Rat IgG2a eF450 (eBR2a), Rat IgG2a FITC (eBR2a), Rat IgG2a PE (eBR2a), Rat IgG2a PE-Cy7 (eBR2a), Rat IgG2a APC (eBR2a), Rat IgG2a BV421 (RTK2758, BioLegend), Rat IgG2a BV650 (RTK2758, BioLegend), Rat IgG2a PerCP-eFluor 710 (eBR2a), Rat IgG2b PE (10H5), Armenian Hamster IgG APC (eBio299Arm), Rat IgG1 Biotin (eBRG1) and Rat IgG1 BV650 (RTK2071; BioLegend).

    Techniques: Mouse Assay, Immunofluorescence, Microscopy, Derivative Assay, Fluorescence

    The non-obese diabetic (NOD) thymus harbors a unique pattern of immunoglobulin (Ig) isotypes. (A) Number of IgM − IgD − IgA + , IgM − IgD − IgE + , and IgM − IgD − IgG + B cells in the thymus of 11- to 14-week-old female B6 ( n = 10) or female NOD mice ( n = 10). (B) Number of IgM − IgD + IgA + , IgM − IgD + IgE + , and IgM − IgD + IgG + B cells in the thymus of 11- to 14-week-old female B6 ( n = 10) and female NOD mice ( n = 10). (C–F) Optical density (OD) values of the respective Igs in cell-free tissue supernatants (C,E) or serum (D,F) . A total of six female B6 and six female NOD mice were assessed in two independent experiments. Data are presented as scatter plot, each dot equating to one mouse and bar representing the mean. P values were calculated using the Mann–Whitney U -test analysis and are shown in this figure; ns, not significant.

    Journal: Frontiers in Immunology

    Article Title: Thymic B Cell-Mediated Attack of Thymic Stroma Precedes Type 1 Diabetes Development

    doi: 10.3389/fimmu.2018.01281

    Figure Lengend Snippet: The non-obese diabetic (NOD) thymus harbors a unique pattern of immunoglobulin (Ig) isotypes. (A) Number of IgM − IgD − IgA + , IgM − IgD − IgE + , and IgM − IgD − IgG + B cells in the thymus of 11- to 14-week-old female B6 ( n = 10) or female NOD mice ( n = 10). (B) Number of IgM − IgD + IgA + , IgM − IgD + IgE + , and IgM − IgD + IgG + B cells in the thymus of 11- to 14-week-old female B6 ( n = 10) and female NOD mice ( n = 10). (C–F) Optical density (OD) values of the respective Igs in cell-free tissue supernatants (C,E) or serum (D,F) . A total of six female B6 and six female NOD mice were assessed in two independent experiments. Data are presented as scatter plot, each dot equating to one mouse and bar representing the mean. P values were calculated using the Mann–Whitney U -test analysis and are shown in this figure; ns, not significant.

    Article Snippet: The gates were defined using fluorescence minus one and isotype controls: Rat IgG2a eF450 (eBR2a), Rat IgG2a FITC (eBR2a), Rat IgG2a PE (eBR2a), Rat IgG2a PE-Cy7 (eBR2a), Rat IgG2a APC (eBR2a), Rat IgG2a BV421 (RTK2758, BioLegend), Rat IgG2a BV650 (RTK2758, BioLegend), Rat IgG2a PerCP-eFluor 710 (eBR2a), Rat IgG2b PE (10H5), Armenian Hamster IgG APC (eBio299Arm), Rat IgG1 Biotin (eBRG1) and Rat IgG1 BV650 (RTK2071; BioLegend).

    Techniques: Mouse Assay, MANN-WHITNEY

    The number of ETPs and TECs was significantly increased in allo-BMT recipients after rIL-7/HGFβ treatment. Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice and treated with cytokines as in Figure 1 . On day 30 after BMT, ETPs and TECs were analyzed. (A) Representative flow cytometric profiles showing the percentage of donor-origin lineage - c-kit + IL-7Rα - CD44 + CD25 - ETPs in total thymocytes. (B) Number of donor-origin ETPs in the cytokine-treated BMT mice. (C) Representative flow cytometric profiles showing the percentage of CD45 - EpCAM + MHC II + Ly51 + cTECs and CD45 - EpCAM + MHC II + Ly51 - mTECs in total TECs of the rIL-7/HGFβ-treated BMT mice. (D) Number of total TECs, cTEC and mTECs in the cytokine-treated BMT mice. The data are representative of 2 independent experiments with 4-6 mice per group. * P

    Journal: PLoS ONE

    Article Title: Recombinant IL-7/HGF? Hybrid Cytokine Enhances T Cell Recovery in Mice Following Allogeneic Bone Marrow Transplantation

    doi: 10.1371/journal.pone.0082998

    Figure Lengend Snippet: The number of ETPs and TECs was significantly increased in allo-BMT recipients after rIL-7/HGFβ treatment. Lethally irradiated BALB/c mice were injected with TCD-BM from B6 mice and treated with cytokines as in Figure 1 . On day 30 after BMT, ETPs and TECs were analyzed. (A) Representative flow cytometric profiles showing the percentage of donor-origin lineage - c-kit + IL-7Rα - CD44 + CD25 - ETPs in total thymocytes. (B) Number of donor-origin ETPs in the cytokine-treated BMT mice. (C) Representative flow cytometric profiles showing the percentage of CD45 - EpCAM + MHC II + Ly51 + cTECs and CD45 - EpCAM + MHC II + Ly51 - mTECs in total TECs of the rIL-7/HGFβ-treated BMT mice. (D) Number of total TECs, cTEC and mTECs in the cytokine-treated BMT mice. The data are representative of 2 independent experiments with 4-6 mice per group. * P

    Article Snippet: Direct or indirect staining of fluorochrome-conjugated antibodies included: CD4, CD8, CD25, CD44, CD62L, c-kit, IL-7Rα, BP-1, CD45, I-A, H-2Kb , CD45.1, Ki67, CD69, EpCAM1, IL-2, IFN-γ, TNFα, Bcl-2, and a panel of TCR Vβ clonotypes (BioLegend, BD Biosciences, San Jose, CA, or eBioscience, San Diego, CA), as well as Bcl-xL (Cell Signaling Technology, Inc., Danvers, MA).

    Techniques: Irradiation, Mouse Assay, Injection, Flow Cytometry