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  • 99
    Thermo Fisher igm invitrogen ab
    Antibody (Ab) profiles of chikungunya virus (CHIKV)–infected patients. A , Virus-specific <t>immunoglobulin</t> M <t>(IgM)</t> and immunoglobulin G (IgG) antibody titers in plasma samples (n = 30) at a dilution of 1:2000 were determined by enzyme-linked immunosorbent assay (ELISA) using purified CHIKV virions. B , Detection of CHIKV by plasma from CHIKV-infected patients. HEK 293T cells were infected with CHIKV (SGP11) at a multiplicity of infection of 10, fixed at 6 h postinfection, and stained with 2 representative patients’ plasma at 2–3 months post–illness onset (PIO) using a dilution of 1:500. Healthy plasma was used as a control. CHIKV antigen was detected by antihuman IgG antibody conjugated to Alexa Fluor 488 (green). 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. Scale bar: 10 μm. C , Virus-specific IgG isotype titers in plasma samples. Immunoglobulin G1 (IgG1) (□), immunoglobulin G2 (IgG2) (Δ), immunoglobulin G3 (IgG3) (•), or immunoglobulin G4 (IgG4) (⋄) Abs were determined as in ( A ) using specific secondary antibodies. Each point represents the mean of 30 patients’ samples from the cohort. D , Chikungunya virion–based ELISA was used to determine virus-specific IgG isotype titers in plasma samples (median, 10 d PIO; n = 30) at a dilution of 1:100. Anti-CHIKV IgG1, IgG2, IgG3, or IgG4 Abs were determined using specific secondary Abs. Data are presented as mean ± standard error of the mean (SEM) of 30 patients’ samples and are representative of 2 independent experiments with similar results. E , Profile of IgG3 levels at different times PIO in early (n = 16) and late IgG3 responders (n = 14) according to the pattern of IgG3 titer at median, 10 d PIO. Data are presented as mean ± SEM. Data are representative of 2 independent experiments with similar results. Statistical significance was measured using Mann–Whitney U test. ** P
    Igm Invitrogen Ab, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore anti igm antibody
    AKT inhibition enhances proximal BCR signaling in GCBCs. a , Purified <t>NBCs</t> and GCBCs from MEG mice were treated with DMSO or AKT inhibitor for 40 min before <t>anti-IgM</t> stimulation for the times indicated. Cell lysates were analyzed by immunoblot. Left panel, representative immunoblot of three independent experiments for the p-Tyr species indicated, with cells pooled from 3 to 5 mice in each experiment; right panel, quantitation of immunoblots from all three experiments, the ratios of pY396/pY507 of LYN and p-SYK/Actin were normalized to DMSO treated NBCs (time 0) as 1. Data are mean ± SEM; P values are comparing treatments (DMSO vs AKT inhibitor) by two-way ANOVA (two factors: treatment and time). (pY396/pY507 NBCs: F=0.005502, d.f.=20; pY396/pY507 GCBCs F=24.32, d.f.=20; p-SYK/Actin NBCs: F=0.3387, d.f.=20; p-SYK/Actin GCBCs: F=9.807, d.f.=20) b , Splenocytes from immunized MEG mice were treated with DMSO or AKT inhibitor for 40 min followed by anti-IgM stimulation for indicated time points. Cells were then analyzed by flow cytometry (n=4 from two independent experiments). Representative histogram (left panel) and statistical analysis for MFI of all samples (right panel) are shown for GCBCs. Data are mean ± SEM; P values are comparing treatments (DMSO vs AKT inhibitor) by two-way ANOVA (two factors: treatment and time). (p-BTK: F=9.034, d.f.=30; p-PLCγ2: F=13.15, d.f.=30).
    Anti Igm Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Pharmingen immunoglobulin m igm antibodies
    AKT inhibition enhances proximal BCR signaling in GCBCs. a , Purified <t>NBCs</t> and GCBCs from MEG mice were treated with DMSO or AKT inhibitor for 40 min before <t>anti-IgM</t> stimulation for the times indicated. Cell lysates were analyzed by immunoblot. Left panel, representative immunoblot of three independent experiments for the p-Tyr species indicated, with cells pooled from 3 to 5 mice in each experiment; right panel, quantitation of immunoblots from all three experiments, the ratios of pY396/pY507 of LYN and p-SYK/Actin were normalized to DMSO treated NBCs (time 0) as 1. Data are mean ± SEM; P values are comparing treatments (DMSO vs AKT inhibitor) by two-way ANOVA (two factors: treatment and time). (pY396/pY507 NBCs: F=0.005502, d.f.=20; pY396/pY507 GCBCs F=24.32, d.f.=20; p-SYK/Actin NBCs: F=0.3387, d.f.=20; p-SYK/Actin GCBCs: F=9.807, d.f.=20) b , Splenocytes from immunized MEG mice were treated with DMSO or AKT inhibitor for 40 min followed by anti-IgM stimulation for indicated time points. Cells were then analyzed by flow cytometry (n=4 from two independent experiments). Representative histogram (left panel) and statistical analysis for MFI of all samples (right panel) are shown for GCBCs. Data are mean ± SEM; P values are comparing treatments (DMSO vs AKT inhibitor) by two-way ANOVA (two factors: treatment and time). (p-BTK: F=9.034, d.f.=30; p-PLCγ2: F=13.15, d.f.=30).
    Immunoglobulin M Igm Antibodies, supplied by Pharmingen, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Jackson Immuno anti igm ab
    TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml <t>anti-CD40</t> and 10 µg/ml <t>anti-IgM</t> either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P
    Anti Igm Ab, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 88/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ICN Biomedicals anti igm ab
    TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml <t>anti-CD40</t> and 10 µg/ml <t>anti-IgM</t> either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P
    Anti Igm Ab, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Immunology Consultant Laboratory immunoglobulin m igm capture antibody
    TGF-β regulates T cell-dependent B cell activation. (A) Histogram plots of CFSE-labeled B220 + B cells stimulated by 10 µg/ml <t>anti-CD40</t> and 10 µg/ml <t>anti-IgM</t> either with TGF-β1 or TGF-β3 for 3 days ( n = 3). Data are representative histograms with CFSE fluorescence gated on B220 + cells and the percentage of proliferating B220 + CFSE − cells. (B) Total IgG and IgA antibody titers in the supernatants of anti-CD40/IL-4 stimulated B cells with either TGF-β1 or TGF-β3 and 50 ng/ml interleukin-10 for 7 days quantified by ELISA ( n = 3). (C) Flow cytometry analyzing the expression of B220 and CD138 in B cells stimulated with anti-CD40/anti-IgM either with or without TGF-β1 or TGF-β3 for 3 days (left), and quantification of B220 + CD138 + plasmablasts (right) ( n = 3). Data are representative of three independent experiments in (A–C) . (D) C57BL/6 (B6) mice treated with indicated pCAGGS plasmid vectors were by i.p. injection of 100 µg NP-KLH in alum. Anti-NP-bovine serum albumin antibody titers in the sera ( n = 16) were quantified by ELISA 7 days after the immunization. (E) Flow cytometric (FCM) plots and quantification of GL-7 + Fas + germinal center B cells in B220 + cells from B6 mice administered the indicated pCAGGS vectors and immunized with 100 µg NP-KLH in alum ( n = 13–14). P
    Immunoglobulin M Igm Capture Antibody, supplied by Immunology Consultant Laboratory, used in various techniques. Bioz Stars score: 88/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad anti igm ab
    The Erk/DNMT pathway is defective in <t>SLE</t> B cells. A–B: Cytoplasmic staining of pErk (A) and DNMT1 (B) in permeabilized B cells stimulated with <t>anti-IgM</t> or not for 10 min (pErk) and 24 h (DNMT1). Representative flow cytometric profiles are represented.
    Anti Igm Ab, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Fuller Laboratories immunoglobulin m igm
    The Erk/DNMT pathway is defective in <t>SLE</t> B cells. A–B: Cytoplasmic staining of pErk (A) and DNMT1 (B) in permeabilized B cells stimulated with <t>anti-IgM</t> or not for 10 min (pErk) and 24 h (DNMT1). Representative flow cytometric profiles are represented.
    Immunoglobulin M Igm, supplied by Fuller Laboratories, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SouthernBiotech anti igm ab m4
    The Erk/DNMT pathway is defective in <t>SLE</t> B cells. A–B: Cytoplasmic staining of pErk (A) and DNMT1 (B) in permeabilized B cells stimulated with <t>anti-IgM</t> or not for 10 min (pErk) and 24 h (DNMT1). Representative flow cytometric profiles are represented.
    Anti Igm Ab M4, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Becton Dickinson immunoglobulin m igm
    Increased levels of serum immunoglobulin G1 (IgG1) in Pten +/– SHIP +/– (phosphatase with tensin homology/SH2-containing inositol phosphatase) mice. (a) Spontaneous increases in circulating serum IgG1 levels were detected in the sera of 8-week-old Pten +/– SHIP +/– mice, as compared to the other genotypes, and this pattern remained steady and elevated over time. (b) Serum levels of circulating <t>immunoglobulin</t> M <t>(IgM)</t> were not significantly increased over all genotypes at the 8-week point, but steadily increased, becoming significant at later time-points. (c) Levels of circulating immunoglobulin G2b (IgG2b) did not achieve statistical significance compared to all genotypes until the 24-week time-point. Values were representative of sera from at least nine animals per genotype, assessed in duplicate. Error bars represent the standard error of the mean (SEM) of the duplicate readings. * P
    Immunoglobulin M Igm, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    SouthernBiotech immunoglobulin m igm
    Bortezomib-mediated long-lived plasma cell (LLPC) depletion is transient. Twelve- to 16-wk-old NZB/W F1 mice were treated with one bortezomib cycle consisting of two bortezomib injections within 36 h and analyzed 12 h and 3, 7 and 15 days after the last bortezomib injection. (A) Representative gating strategy for plasma cell analysis by flow cytometry. Lymphocytes were gated according to their position in forward and sideward scatter, and doublets were excluded from the analysis (not shown). Plasma cells were identified as CD138+ and intracellular kappa light chain + cells both in spleen and bone marrow. Within the plasma cells population, plasmablasts and newly generated plasma cells were identified as cells with a higher expression of MHC-II. Conversely, MHC-II low plasma cells were regarded as LLPCs. A representative plot of the expression of MHC-II on the plasma cell population is shown for spleen and bone marrow 15 days after bortezomib treatment. (B) Absolute numbers of all CD138+/intracellular kappa light chain + plasma cells (white bars) and the subgroups of short-lived, MHC high (gray bars) and long-lived, MHC-II low (black bars) plasma cells in spleens (left) and bone marrows (right) 12 h (0.5 days), 3, 7 and 15 days after one cycle of bortezomib measured by flow cytometry. n = 7 mice per time point. (C) Frequencies of the remaining anti-double-stranded DNA (dsDNA) antibody-secreting cells (ASCs) (immunoglobulin G (IgG) and <t>immunoglobulin</t> M <t>(IgM)</t> combined) in spleen (left) and bone marrow (right) calculated by comparison with untreated mice (day 0), as detected by enzyme-linked immunospot (ELISPOT). n = 4 mice per time point. Data are presented as mean and standard error of the mean (SEM). n.s., nonsignificant. P > 0.05, * P ≤0.05, ** P ≤0.01 by two-tailed unpaired t test.
    Immunoglobulin M Igm, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 91/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam immunoglobulin m igm
    Validation of selected candidate biomarkers for HCC development in a larger patient population. (A) Quantification of plasma concentration of CPN (pg/mg plasma protein), CP, <t>IgM,</t> PON1 (μg/mg plasma protein), C4a (ng/mg plasma protein) and FGA (μg/mg plasma protein) by ELISA. The sample size was 24 in the control group and 22 in the tumor group. * Statistically significant differences between groups. Data as mean ± standard error; (B) ROC curves showing that the best predictive ability to identify patients with HCC belongs to the combination of C4a, FGA, CP and PON1.
    Immunoglobulin M Igm, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam igm anti lewis ab
    <t>Toso/FcμR</t> binds <t>IgM</t>
    Igm Anti Lewis Ab, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SouthernBiotech polyclonal igm capture ab
    <t>Toso/FcμR</t> binds <t>IgM</t>
    Polyclonal Igm Capture Ab, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 85/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Focus Diagnostics Inc immunoglobulin m igm
    <t>Toso/FcμR</t> binds <t>IgM</t>
    Immunoglobulin M Igm, supplied by Focus Diagnostics Inc, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    The Jackson Laboratory anti immunoglobulin m anti igm antibody
    GDC-0853 represses NF-κB–dependent gene transcription. CLL patient cells were treated with 1 µM BTK inhibitor for 72 hours with or without <t>anti-IgM</t> stimulation and assessed for gene expression changes of NF-κB targets by RT-PCR (N = 8). ** P ≤ .01; *** P ≤ .001.
    Anti Immunoglobulin M Anti Igm Antibody, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    SouthernBiotech goat anti human immunoglobulin m igm f ab 2
    GDC-0853 represses NF-κB–dependent gene transcription. CLL patient cells were treated with 1 µM BTK inhibitor for 72 hours with or without <t>anti-IgM</t> stimulation and assessed for gene expression changes of NF-κB targets by RT-PCR (N = 8). ** P ≤ .01; *** P ≤ .001.
    Goat Anti Human Immunoglobulin M Igm F Ab 2, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Bio-Synthesis Inc anti mouse igm ab
    Dynamics of humoral responses in mice after orally infected with T . gondii cysts. Detection of T . gondii -specific <t>IgG</t> (A) and <t>IgM</t> (B) in the serum of BALB/c mice (n = 3) infected with four cysts at 1, 3, 5, 7, 9, 11, 13, 15, 22, 30, 45 dpi with ELISA. Results are expressed as mean of the OD 450/630 ± S.D. Inverted black triangle p
    Anti Mouse Igm Ab, supplied by Bio-Synthesis Inc, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Antibody (Ab) profiles of chikungunya virus (CHIKV)–infected patients. A , Virus-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibody titers in plasma samples (n = 30) at a dilution of 1:2000 were determined by enzyme-linked immunosorbent assay (ELISA) using purified CHIKV virions. B , Detection of CHIKV by plasma from CHIKV-infected patients. HEK 293T cells were infected with CHIKV (SGP11) at a multiplicity of infection of 10, fixed at 6 h postinfection, and stained with 2 representative patients’ plasma at 2–3 months post–illness onset (PIO) using a dilution of 1:500. Healthy plasma was used as a control. CHIKV antigen was detected by antihuman IgG antibody conjugated to Alexa Fluor 488 (green). 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. Scale bar: 10 μm. C , Virus-specific IgG isotype titers in plasma samples. Immunoglobulin G1 (IgG1) (□), immunoglobulin G2 (IgG2) (Δ), immunoglobulin G3 (IgG3) (•), or immunoglobulin G4 (IgG4) (⋄) Abs were determined as in ( A ) using specific secondary antibodies. Each point represents the mean of 30 patients’ samples from the cohort. D , Chikungunya virion–based ELISA was used to determine virus-specific IgG isotype titers in plasma samples (median, 10 d PIO; n = 30) at a dilution of 1:100. Anti-CHIKV IgG1, IgG2, IgG3, or IgG4 Abs were determined using specific secondary Abs. Data are presented as mean ± standard error of the mean (SEM) of 30 patients’ samples and are representative of 2 independent experiments with similar results. E , Profile of IgG3 levels at different times PIO in early (n = 16) and late IgG3 responders (n = 14) according to the pattern of IgG3 titer at median, 10 d PIO. Data are presented as mean ± SEM. Data are representative of 2 independent experiments with similar results. Statistical significance was measured using Mann–Whitney U test. ** P

    Journal: The Journal of Infectious Diseases

    Article Title: Early Appearance of Neutralizing Immunoglobulin G3 Antibodies Is Associated With Chikungunya Virus Clearance and Long-term Clinical Protection

    doi: 10.1093/infdis/jis033

    Figure Lengend Snippet: Antibody (Ab) profiles of chikungunya virus (CHIKV)–infected patients. A , Virus-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibody titers in plasma samples (n = 30) at a dilution of 1:2000 were determined by enzyme-linked immunosorbent assay (ELISA) using purified CHIKV virions. B , Detection of CHIKV by plasma from CHIKV-infected patients. HEK 293T cells were infected with CHIKV (SGP11) at a multiplicity of infection of 10, fixed at 6 h postinfection, and stained with 2 representative patients’ plasma at 2–3 months post–illness onset (PIO) using a dilution of 1:500. Healthy plasma was used as a control. CHIKV antigen was detected by antihuman IgG antibody conjugated to Alexa Fluor 488 (green). 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. Scale bar: 10 μm. C , Virus-specific IgG isotype titers in plasma samples. Immunoglobulin G1 (IgG1) (□), immunoglobulin G2 (IgG2) (Δ), immunoglobulin G3 (IgG3) (•), or immunoglobulin G4 (IgG4) (⋄) Abs were determined as in ( A ) using specific secondary antibodies. Each point represents the mean of 30 patients’ samples from the cohort. D , Chikungunya virion–based ELISA was used to determine virus-specific IgG isotype titers in plasma samples (median, 10 d PIO; n = 30) at a dilution of 1:100. Anti-CHIKV IgG1, IgG2, IgG3, or IgG4 Abs were determined using specific secondary Abs. Data are presented as mean ± standard error of the mean (SEM) of 30 patients’ samples and are representative of 2 independent experiments with similar results. E , Profile of IgG3 levels at different times PIO in early (n = 16) and late IgG3 responders (n = 14) according to the pattern of IgG3 titer at median, 10 d PIO. Data are presented as mean ± SEM. Data are representative of 2 independent experiments with similar results. Statistical significance was measured using Mann–Whitney U test. ** P

    Article Snippet: Horseradish peroxidase–conjugated mouse antihuman immunoglobulin G (IgG), immunoglobulin G1 (IgG1), immunoglobulin G2 (IgG2), IgG3, immunoglobulin G4 (IgG4), and immunoglobulin M (IgM) (Molecular Probes) were used to detect human antibodies bound to virus-coated wells.

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Purification, Staining, MANN-WHITNEY

    Relationship among EBV status, form of EBV latency, and susceptibility to apoptosis in Awia-BL cells. ( A and B ) Mean results from multiple apoptosis assays in which Awia-BL clones were exposed to anti-IgM at 10 μg/ml for 72 h ( A ) or ionomycin at 1 μg/ml for 48 h ( B ) and then stained with propidium iodide and Syto 16. Results are expressed as the percentage of cells induced to die. Extending the assay period up to 96 h in each case did not affect the pattern of results (data not shown). ( C ) Mean results from additional assays in which EBV-negative and Latency I clones were tested at lower doses of 2.5 μg/ml anti-IgM and 0.25 μg/ml ionomycin. Note that in all experiments, essentially similar results were obtained by using caspase cleavage as a marker of apoptosis in single cells.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Three restricted forms of Epstein-Barr virus latency counteracting apoptosis in c-myc-expressing Burkitt lymphoma cells

    doi: 10.1073/pnas.0509988103

    Figure Lengend Snippet: Relationship among EBV status, form of EBV latency, and susceptibility to apoptosis in Awia-BL cells. ( A and B ) Mean results from multiple apoptosis assays in which Awia-BL clones were exposed to anti-IgM at 10 μg/ml for 72 h ( A ) or ionomycin at 1 μg/ml for 48 h ( B ) and then stained with propidium iodide and Syto 16. Results are expressed as the percentage of cells induced to die. Extending the assay period up to 96 h in each case did not affect the pattern of results (data not shown). ( C ) Mean results from additional assays in which EBV-negative and Latency I clones were tested at lower doses of 2.5 μg/ml anti-IgM and 0.25 μg/ml ionomycin. Note that in all experiments, essentially similar results were obtained by using caspase cleavage as a marker of apoptosis in single cells.

    Article Snippet: BL cells at 3 × 105 cells per ml were exposed at 37°C to known concentrations of ionomycin (Sigma) or anti-IgM antibody (ICN Flow) for 24, 48, 72, and 96 h, then dually stained with 25 nM Syto 16 (Molecular Probes Europe, Leiden, The Netherlands) and 2.5 μg/ml propidium iodide (Sigma) and analyzed immediately by flow cytometry as described in refs. and .

    Techniques: Clone Assay, Staining, Marker

    AKT inhibition enhances proximal BCR signaling in GCBCs. a , Purified NBCs and GCBCs from MEG mice were treated with DMSO or AKT inhibitor for 40 min before anti-IgM stimulation for the times indicated. Cell lysates were analyzed by immunoblot. Left panel, representative immunoblot of three independent experiments for the p-Tyr species indicated, with cells pooled from 3 to 5 mice in each experiment; right panel, quantitation of immunoblots from all three experiments, the ratios of pY396/pY507 of LYN and p-SYK/Actin were normalized to DMSO treated NBCs (time 0) as 1. Data are mean ± SEM; P values are comparing treatments (DMSO vs AKT inhibitor) by two-way ANOVA (two factors: treatment and time). (pY396/pY507 NBCs: F=0.005502, d.f.=20; pY396/pY507 GCBCs F=24.32, d.f.=20; p-SYK/Actin NBCs: F=0.3387, d.f.=20; p-SYK/Actin GCBCs: F=9.807, d.f.=20) b , Splenocytes from immunized MEG mice were treated with DMSO or AKT inhibitor for 40 min followed by anti-IgM stimulation for indicated time points. Cells were then analyzed by flow cytometry (n=4 from two independent experiments). Representative histogram (left panel) and statistical analysis for MFI of all samples (right panel) are shown for GCBCs. Data are mean ± SEM; P values are comparing treatments (DMSO vs AKT inhibitor) by two-way ANOVA (two factors: treatment and time). (p-BTK: F=9.034, d.f.=30; p-PLCγ2: F=13.15, d.f.=30).

    Journal: Nature immunology

    Article Title: The AKT kinase signaling network is rewired by PTEN to control proximal BCR signaling in germinal center B cells

    doi: 10.1038/s41590-019-0376-3

    Figure Lengend Snippet: AKT inhibition enhances proximal BCR signaling in GCBCs. a , Purified NBCs and GCBCs from MEG mice were treated with DMSO or AKT inhibitor for 40 min before anti-IgM stimulation for the times indicated. Cell lysates were analyzed by immunoblot. Left panel, representative immunoblot of three independent experiments for the p-Tyr species indicated, with cells pooled from 3 to 5 mice in each experiment; right panel, quantitation of immunoblots from all three experiments, the ratios of pY396/pY507 of LYN and p-SYK/Actin were normalized to DMSO treated NBCs (time 0) as 1. Data are mean ± SEM; P values are comparing treatments (DMSO vs AKT inhibitor) by two-way ANOVA (two factors: treatment and time). (pY396/pY507 NBCs: F=0.005502, d.f.=20; pY396/pY507 GCBCs F=24.32, d.f.=20; p-SYK/Actin NBCs: F=0.3387, d.f.=20; p-SYK/Actin GCBCs: F=9.807, d.f.=20) b , Splenocytes from immunized MEG mice were treated with DMSO or AKT inhibitor for 40 min followed by anti-IgM stimulation for indicated time points. Cells were then analyzed by flow cytometry (n=4 from two independent experiments). Representative histogram (left panel) and statistical analysis for MFI of all samples (right panel) are shown for GCBCs. Data are mean ± SEM; P values are comparing treatments (DMSO vs AKT inhibitor) by two-way ANOVA (two factors: treatment and time). (p-BTK: F=9.034, d.f.=30; p-PLCγ2: F=13.15, d.f.=30).

    Article Snippet: NBCs or GCBCs were activated with anti-IgM antibody in the presence or absence of 10 μM AKT1/2 kinase inhibitor (Calbiochem).

    Techniques: Inhibition, Purification, Mouse Assay, Quantitation Assay, Western Blot, Flow Cytometry, Cytometry

    PTEN is highly expressed in GCBCs, controlling phosphatidylinositol phosphate generation and restraining AKT S473 phosphorylation. a , Protein expression was analyzed by immunoblot with freshly purified B1–8i NBCs and GCBCs; lower graph shows the quantitation of immunoblot data with the mean value. The ratio of PTEN/Actin was normalized to NBCs as 1. (n=3 from three independent experiments, each sample contain cells pooled from two mice). P values, two-tailed unpaired Student’s t -test (t=4.161, d.f.=4). b , Purified MEG NBCs and GCBCs were stimulated with anti-IgM. Inositol lipids from cell lysates were measured by ELISA. Symbols are means and error bars are SEM from two independent experiments with cells pooled from five mice per group in each experiment; P values: two-way ANOVA with Sidak’s multiple comparisons test (PtdIns(3,4,5)P3: F= 44.76, d.f.=4; PtdIns(3,4)P2: F=33.68, d.f.=4; PtdIns(4,5)P2: F=11.31, d.f.=4). c , NBCs and GCBCs from MEG mice (n=2 except “no inhibitor” condition at 0, 1, and 5 minutes, where n=3)) were treated with the PTEN inhibitor (SF1670) for 10 minutes and then stimulated with anti-IgM. Inositol lipid species were analyzed by ELISA. Cells were pooled from five mice in each experiment. Symbols are means and error bars are SEM. P values compare DMSO and SF1670 treated samples for NBCs (red) and GCBCs (blue) by two-way ANOVA with Sidak’s multiple comparisons test (PtdIns(3,4,5)P3: F=76.71, d.f.=3; PtdIns(3,4)P2: F=214.4, d.f.=3; PtdIns(4,5)P2: F=88.16, d.f.=3). d , B1–8i splenocytes were treated with DMSO or SF1670 for 30 minutes before BCR stimulation with NP-Ficoll. Cells were fixed and analyzed by flow cytometry 5 min (p-AKT) or 20 min (p-S6) post stimulation. Right: fold change of MFI, normalized to DMSO-alone treated samples. Data are mean ± SEM from five mice from two independent experiments; P values, one-way ANOVA followed by Turkey’s multiple comparisons test (p-AKT NBCs: F=71.93, d.f.=16; p-AKT GCBCs: F=262.2, d.f.=16; p-S6 NBCs: F=64.86, d.f.=16; p-S6 GCBCs: F=98.72, d.f.=16).

    Journal: Nature immunology

    Article Title: The AKT kinase signaling network is rewired by PTEN to control proximal BCR signaling in germinal center B cells

    doi: 10.1038/s41590-019-0376-3

    Figure Lengend Snippet: PTEN is highly expressed in GCBCs, controlling phosphatidylinositol phosphate generation and restraining AKT S473 phosphorylation. a , Protein expression was analyzed by immunoblot with freshly purified B1–8i NBCs and GCBCs; lower graph shows the quantitation of immunoblot data with the mean value. The ratio of PTEN/Actin was normalized to NBCs as 1. (n=3 from three independent experiments, each sample contain cells pooled from two mice). P values, two-tailed unpaired Student’s t -test (t=4.161, d.f.=4). b , Purified MEG NBCs and GCBCs were stimulated with anti-IgM. Inositol lipids from cell lysates were measured by ELISA. Symbols are means and error bars are SEM from two independent experiments with cells pooled from five mice per group in each experiment; P values: two-way ANOVA with Sidak’s multiple comparisons test (PtdIns(3,4,5)P3: F= 44.76, d.f.=4; PtdIns(3,4)P2: F=33.68, d.f.=4; PtdIns(4,5)P2: F=11.31, d.f.=4). c , NBCs and GCBCs from MEG mice (n=2 except “no inhibitor” condition at 0, 1, and 5 minutes, where n=3)) were treated with the PTEN inhibitor (SF1670) for 10 minutes and then stimulated with anti-IgM. Inositol lipid species were analyzed by ELISA. Cells were pooled from five mice in each experiment. Symbols are means and error bars are SEM. P values compare DMSO and SF1670 treated samples for NBCs (red) and GCBCs (blue) by two-way ANOVA with Sidak’s multiple comparisons test (PtdIns(3,4,5)P3: F=76.71, d.f.=3; PtdIns(3,4)P2: F=214.4, d.f.=3; PtdIns(4,5)P2: F=88.16, d.f.=3). d , B1–8i splenocytes were treated with DMSO or SF1670 for 30 minutes before BCR stimulation with NP-Ficoll. Cells were fixed and analyzed by flow cytometry 5 min (p-AKT) or 20 min (p-S6) post stimulation. Right: fold change of MFI, normalized to DMSO-alone treated samples. Data are mean ± SEM from five mice from two independent experiments; P values, one-way ANOVA followed by Turkey’s multiple comparisons test (p-AKT NBCs: F=71.93, d.f.=16; p-AKT GCBCs: F=262.2, d.f.=16; p-S6 NBCs: F=64.86, d.f.=16; p-S6 GCBCs: F=98.72, d.f.=16).

    Article Snippet: NBCs or GCBCs were activated with anti-IgM antibody in the presence or absence of 10 μM AKT1/2 kinase inhibitor (Calbiochem).

    Techniques: Expressing, Purification, Quantitation Assay, Mouse Assay, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    Phosphorylation of AKT is altered in GCBCs compared to NBCs. a , AKT protein expression and phosphorylation was analyzed by immunoblot with freshly purified B1–8i NBCs and GCBCs (n=2); lower graph shows the quantitation of bands, with bars showing means of replicates. The ratios were normalized to those of NBCs, given a value of 1. b , Splenocytes from immunized MEG mice (n=3) were instantly fixed in 1.5% PFA and then analyzed by flow cytometry; lower graph shows fold change of median fluorescence intensity (MFI) and was normalized to NBCs as 1. Graph shows the quantitation as in (b). c , Western blot for p-PDK1(S241) and total PKD1 in purified MEG NBCs and GCBCs (n=2); lower graphs show the quantitation of bands as in (b). The ratios were normalized to those of NBCs, given a value of 1. d , Total splenocytes from immunized MEG mice (n=3) were stimulated with anti-IgM for indicated time points and p-AKT in GCBCs and NBCs was analyzed by flow cytometry. e , Purified MEG NBCs and GCBCs (n=2) were rested for 40 minutes and stimulated with anti-IgM for indicated time points and cell lysates were analyzed by immunoblot. Data represent two independent experiments with cells pooled from two mice per group in each experiment ( a,c,e ) or three independent experiments with one mouse tested in each experiment ( b,d ). P values, two-tailed unpaired Student’s t -test ( a,b,c ). (p-AKT(T308)/Actin: t=6.676, d.f.=2; p-AKT(T308)/total AKT: t=18.67, d.f.=2; total AKT/Actin: t=11.50, df=2; p-AKT(T308) normalized MFI: t=8.758, d.f.=4; p-PDK1/Actin: t=5.830, df=2; total PDK1/Actin: t=4.609, df=2).

    Journal: Nature immunology

    Article Title: The AKT kinase signaling network is rewired by PTEN to control proximal BCR signaling in germinal center B cells

    doi: 10.1038/s41590-019-0376-3

    Figure Lengend Snippet: Phosphorylation of AKT is altered in GCBCs compared to NBCs. a , AKT protein expression and phosphorylation was analyzed by immunoblot with freshly purified B1–8i NBCs and GCBCs (n=2); lower graph shows the quantitation of bands, with bars showing means of replicates. The ratios were normalized to those of NBCs, given a value of 1. b , Splenocytes from immunized MEG mice (n=3) were instantly fixed in 1.5% PFA and then analyzed by flow cytometry; lower graph shows fold change of median fluorescence intensity (MFI) and was normalized to NBCs as 1. Graph shows the quantitation as in (b). c , Western blot for p-PDK1(S241) and total PKD1 in purified MEG NBCs and GCBCs (n=2); lower graphs show the quantitation of bands as in (b). The ratios were normalized to those of NBCs, given a value of 1. d , Total splenocytes from immunized MEG mice (n=3) were stimulated with anti-IgM for indicated time points and p-AKT in GCBCs and NBCs was analyzed by flow cytometry. e , Purified MEG NBCs and GCBCs (n=2) were rested for 40 minutes and stimulated with anti-IgM for indicated time points and cell lysates were analyzed by immunoblot. Data represent two independent experiments with cells pooled from two mice per group in each experiment ( a,c,e ) or three independent experiments with one mouse tested in each experiment ( b,d ). P values, two-tailed unpaired Student’s t -test ( a,b,c ). (p-AKT(T308)/Actin: t=6.676, d.f.=2; p-AKT(T308)/total AKT: t=18.67, d.f.=2; total AKT/Actin: t=11.50, df=2; p-AKT(T308) normalized MFI: t=8.758, d.f.=4; p-PDK1/Actin: t=5.830, df=2; total PDK1/Actin: t=4.609, df=2).

    Article Snippet: NBCs or GCBCs were activated with anti-IgM antibody in the presence or absence of 10 μM AKT1/2 kinase inhibitor (Calbiochem).

    Techniques: Expressing, Purification, Quantitation Assay, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence, Western Blot, Two Tailed Test

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

    Journal: Frontiers in Immunology

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

    doi: 10.3389/fimmu.2018.01364

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

    Article Snippet: Cell Proliferation Assay Isolated splenic B cells were labeled with 0.2 µg/ml 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE; Dojindo, Kumamoto, Japan) at 37°C for 10 min, and then 3 × 105 B cells/well in 96-well flat bottom plate were stimulated by 10 µg/ml anti-CD40 Ab and 10 µg/ml anti-IgM Ab (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) or 10 µg/ml LPS with 2 ng/ml TGF-β3 and/or 50 ng/ml IL-10 for 3 days.

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

    Plasma levels of antigen-specific antibody. Homologous vaccines were used as Ags to coat the plates. Plasma samples from controls (circles) and patients (stars) were diluted and tested. Median concentrations of vaccine Ag-specific IgG (A), IgM (B), and

    Journal: Vaccine

    Article Title: Key Differences in B cell Activation Patterns and Immune Correlates among Treated HIV-Infected Patients Versus Healthy Controls Following Influenza Vaccination

    doi: 10.1016/j.vaccine.2015.12.038

    Figure Lengend Snippet: Plasma levels of antigen-specific antibody. Homologous vaccines were used as Ags to coat the plates. Plasma samples from controls (circles) and patients (stars) were diluted and tested. Median concentrations of vaccine Ag-specific IgG (A), IgM (B), and

    Article Snippet: After blocking, freshly isolated B cells (0.5 million cells/well) were added and cultured overnight at 37°C, and the plates were incubated with a biotinylated mouse anti-human IgA, IgG or IgM Fc Ab (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 2 h. Finally, the plates were incubated with alkaline phosphatase-conjugated streptavidin and developed with substrate.

    Techniques:

    T-oligo treatment does not cause apoptosis in normal primary B or T cells. ( a ) Primary B-2 cells were purified from mouse spleens, cultured and stimulated with 1 µg/ml goat anti-mouse IgM F(ab′) 2 antibody (μ chain specific), 1

    Journal: International journal of cancer. Journal international du cancer

    Article Title: Telomere homolog oligonucleotides induce apoptosis in malignant but not in normal lymphoid cells: Mechanism and therapeutic potential

    doi: 10.1002/ijc.23946

    Figure Lengend Snippet: T-oligo treatment does not cause apoptosis in normal primary B or T cells. ( a ) Primary B-2 cells were purified from mouse spleens, cultured and stimulated with 1 µg/ml goat anti-mouse IgM F(ab′) 2 antibody (μ chain specific), 1

    Article Snippet: Primary B cells were isolated from normal mouse spleens by anti-CD43 negative selection with magnetic beads (21; Miltenyi Biotec, Auburn, CA) and stimulated with anti-immunoglobulin M (IgM) (Jackson ImmunoResearch, West Grove, PA) and anti-CD40 antibodies (BD Pharmingen, San Diego, CA) and interleukin-4 (eBioscience, San Diego, CA).

    Techniques: Purification, Cell Culture

    The Erk/DNMT pathway is defective in SLE B cells. A–B: Cytoplasmic staining of pErk (A) and DNMT1 (B) in permeabilized B cells stimulated with anti-IgM or not for 10 min (pErk) and 24 h (DNMT1). Representative flow cytometric profiles are represented.

    Journal: Autoimmunity

    Article Title: DNA methylation modulates HRES1/p28 expression in B cells from patients with Lupus

    doi: 10.3109/08916934.2013.826207

    Figure Lengend Snippet: The Erk/DNMT pathway is defective in SLE B cells. A–B: Cytoplasmic staining of pErk (A) and DNMT1 (B) in permeabilized B cells stimulated with anti-IgM or not for 10 min (pErk) and 24 h (DNMT1). Representative flow cytometric profiles are represented.

    Article Snippet: To test this hypothesis, SLE B cells and HC B cells were stimulated with an anti-IgM Ab and the intracellular phosphorylation status of the MAPK Erk was evaluated after 10 min by flow cytometry.

    Techniques: Staining, Flow Cytometry

    The effect of IL-6 on HRES-1/P28 expression and HRES-1 promoter methylation status. A/C: HRES-1 promoter analysis by MS-PCR in SLE B cells (A) and healthy control B cells (C) after BCR engagement using an anti-IgM Ab. Based on our previous observation

    Journal: Autoimmunity

    Article Title: DNA methylation modulates HRES1/p28 expression in B cells from patients with Lupus

    doi: 10.3109/08916934.2013.826207

    Figure Lengend Snippet: The effect of IL-6 on HRES-1/P28 expression and HRES-1 promoter methylation status. A/C: HRES-1 promoter analysis by MS-PCR in SLE B cells (A) and healthy control B cells (C) after BCR engagement using an anti-IgM Ab. Based on our previous observation

    Article Snippet: To test this hypothesis, SLE B cells and HC B cells were stimulated with an anti-IgM Ab and the intracellular phosphorylation status of the MAPK Erk was evaluated after 10 min by flow cytometry.

    Techniques: Expressing, Methylation, Mass Spectrometry, Polymerase Chain Reaction

    Increased levels of serum immunoglobulin G1 (IgG1) in Pten +/– SHIP +/– (phosphatase with tensin homology/SH2-containing inositol phosphatase) mice. (a) Spontaneous increases in circulating serum IgG1 levels were detected in the sera of 8-week-old Pten +/– SHIP +/– mice, as compared to the other genotypes, and this pattern remained steady and elevated over time. (b) Serum levels of circulating immunoglobulin M (IgM) were not significantly increased over all genotypes at the 8-week point, but steadily increased, becoming significant at later time-points. (c) Levels of circulating immunoglobulin G2b (IgG2b) did not achieve statistical significance compared to all genotypes until the 24-week time-point. Values were representative of sera from at least nine animals per genotype, assessed in duplicate. Error bars represent the standard error of the mean (SEM) of the duplicate readings. * P

    Journal: Immunology

    Article Title: Compound heterozygosity for Pten and SHIP augments T-dependent humoral immune responses and cytokine production by CD4+ T cells

    doi: 10.1111/j.1365-2567.2004.01901.x

    Figure Lengend Snippet: Increased levels of serum immunoglobulin G1 (IgG1) in Pten +/– SHIP +/– (phosphatase with tensin homology/SH2-containing inositol phosphatase) mice. (a) Spontaneous increases in circulating serum IgG1 levels were detected in the sera of 8-week-old Pten +/– SHIP +/– mice, as compared to the other genotypes, and this pattern remained steady and elevated over time. (b) Serum levels of circulating immunoglobulin M (IgM) were not significantly increased over all genotypes at the 8-week point, but steadily increased, becoming significant at later time-points. (c) Levels of circulating immunoglobulin G2b (IgG2b) did not achieve statistical significance compared to all genotypes until the 24-week time-point. Values were representative of sera from at least nine animals per genotype, assessed in duplicate. Error bars represent the standard error of the mean (SEM) of the duplicate readings. * P

    Article Snippet: ELISA quantification of serum immunoglobulin levels was performed, as previously described, using capture and biotinylated detection antibodies for immunoglobulin G1 (IgG1), immunoglobulin G2b (IgG2b) and immunoglobulin M (IgM) (BD Pharmingen).

    Techniques: Mouse Assay

    Pten +/– SHIP +/– (phosphatase with tensin homology/SH2-containing inositol phosphatase) mice show an increased humoral response to the T-dependent antigen, nitrophenylacetyl (NP)-keyhole limpet haemocyanin (NP-KLH). Serum antibodies against NP-bovine serum albumin (NP-BSA) were assessed from six or seven mice of each genotype that had been challenged with NP-Ficoll or NP-KLH. Anti-NP titres [immunoglobulin M (IgM) and pan immunoglobulin G (IgG)] were determined by enzyme-linked immunosorbent assay (ELISA) on serially diluted serum samples. Bars indicate the average titre, and triangles represent individual mice. No differences were observed in either the IgM or IgG response to NP-Ficoll (a, b). However, statistically significant differences in IgM and IgG responses to NP-KLH were detected (c, d). * P

    Journal: Immunology

    Article Title: Compound heterozygosity for Pten and SHIP augments T-dependent humoral immune responses and cytokine production by CD4+ T cells

    doi: 10.1111/j.1365-2567.2004.01901.x

    Figure Lengend Snippet: Pten +/– SHIP +/– (phosphatase with tensin homology/SH2-containing inositol phosphatase) mice show an increased humoral response to the T-dependent antigen, nitrophenylacetyl (NP)-keyhole limpet haemocyanin (NP-KLH). Serum antibodies against NP-bovine serum albumin (NP-BSA) were assessed from six or seven mice of each genotype that had been challenged with NP-Ficoll or NP-KLH. Anti-NP titres [immunoglobulin M (IgM) and pan immunoglobulin G (IgG)] were determined by enzyme-linked immunosorbent assay (ELISA) on serially diluted serum samples. Bars indicate the average titre, and triangles represent individual mice. No differences were observed in either the IgM or IgG response to NP-Ficoll (a, b). However, statistically significant differences in IgM and IgG responses to NP-KLH were detected (c, d). * P

    Article Snippet: ELISA quantification of serum immunoglobulin levels was performed, as previously described, using capture and biotinylated detection antibodies for immunoglobulin G1 (IgG1), immunoglobulin G2b (IgG2b) and immunoglobulin M (IgM) (BD Pharmingen).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Bortezomib-mediated long-lived plasma cell (LLPC) depletion is transient. Twelve- to 16-wk-old NZB/W F1 mice were treated with one bortezomib cycle consisting of two bortezomib injections within 36 h and analyzed 12 h and 3, 7 and 15 days after the last bortezomib injection. (A) Representative gating strategy for plasma cell analysis by flow cytometry. Lymphocytes were gated according to their position in forward and sideward scatter, and doublets were excluded from the analysis (not shown). Plasma cells were identified as CD138+ and intracellular kappa light chain + cells both in spleen and bone marrow. Within the plasma cells population, plasmablasts and newly generated plasma cells were identified as cells with a higher expression of MHC-II. Conversely, MHC-II low plasma cells were regarded as LLPCs. A representative plot of the expression of MHC-II on the plasma cell population is shown for spleen and bone marrow 15 days after bortezomib treatment. (B) Absolute numbers of all CD138+/intracellular kappa light chain + plasma cells (white bars) and the subgroups of short-lived, MHC high (gray bars) and long-lived, MHC-II low (black bars) plasma cells in spleens (left) and bone marrows (right) 12 h (0.5 days), 3, 7 and 15 days after one cycle of bortezomib measured by flow cytometry. n = 7 mice per time point. (C) Frequencies of the remaining anti-double-stranded DNA (dsDNA) antibody-secreting cells (ASCs) (immunoglobulin G (IgG) and immunoglobulin M (IgM) combined) in spleen (left) and bone marrow (right) calculated by comparison with untreated mice (day 0), as detected by enzyme-linked immunospot (ELISPOT). n = 4 mice per time point. Data are presented as mean and standard error of the mean (SEM). n.s., nonsignificant. P > 0.05, * P ≤0.05, ** P ≤0.01 by two-tailed unpaired t test.

    Journal: Arthritis Research & Therapy

    Article Title: Long-lived plasma cells are early and constantly generated in New Zealand Black/New Zealand White F1 mice and their therapeutic depletion requires a combined targeting of autoreactive plasma cells and their precursors

    doi: 10.1186/s13075-015-0551-3

    Figure Lengend Snippet: Bortezomib-mediated long-lived plasma cell (LLPC) depletion is transient. Twelve- to 16-wk-old NZB/W F1 mice were treated with one bortezomib cycle consisting of two bortezomib injections within 36 h and analyzed 12 h and 3, 7 and 15 days after the last bortezomib injection. (A) Representative gating strategy for plasma cell analysis by flow cytometry. Lymphocytes were gated according to their position in forward and sideward scatter, and doublets were excluded from the analysis (not shown). Plasma cells were identified as CD138+ and intracellular kappa light chain + cells both in spleen and bone marrow. Within the plasma cells population, plasmablasts and newly generated plasma cells were identified as cells with a higher expression of MHC-II. Conversely, MHC-II low plasma cells were regarded as LLPCs. A representative plot of the expression of MHC-II on the plasma cell population is shown for spleen and bone marrow 15 days after bortezomib treatment. (B) Absolute numbers of all CD138+/intracellular kappa light chain + plasma cells (white bars) and the subgroups of short-lived, MHC high (gray bars) and long-lived, MHC-II low (black bars) plasma cells in spleens (left) and bone marrows (right) 12 h (0.5 days), 3, 7 and 15 days after one cycle of bortezomib measured by flow cytometry. n = 7 mice per time point. (C) Frequencies of the remaining anti-double-stranded DNA (dsDNA) antibody-secreting cells (ASCs) (immunoglobulin G (IgG) and immunoglobulin M (IgM) combined) in spleen (left) and bone marrow (right) calculated by comparison with untreated mice (day 0), as detected by enzyme-linked immunospot (ELISPOT). n = 4 mice per time point. Data are presented as mean and standard error of the mean (SEM). n.s., nonsignificant. P > 0.05, * P ≤0.05, ** P ≤0.01 by two-tailed unpaired t test.

    Article Snippet: After extensive washing, plates were incubated with biotin-labeled goat anti-mouse immunoglobulin G (IgG) and immunoglobulin M (IgM) (1 μg/ml, Southern Biotech, Birmingham, AL, USA) for 1 h and followed by ExtrAvidin™-alkaline phosphatase (Sigma-Aldrich) for 20 min.

    Techniques: Mouse Assay, Injection, Flow Cytometry, Cytometry, Generated, Expressing, Enzyme-linked Immunospot, Two Tailed Test

    DOT1L-deficient B cells fail to mount an efficient immune response but show signs of plasma-cell differentiation upon challenge. a) Representative flow cytometry plots of splenic GC B cells 14 days after LCMV Armstrong infection in WT and Dot1L -KO mice and their statistical analyses. Error bars indicate mean with ± SD. b) Representative flow cytometry plots of plasma cells B from the spleen at 14 days after LCMV infection and their statistical analyses. Error bars indicate mean with ± SD. c) LCMV-specific IgM and IgG titers in the serum of WT and KO mice before (D0) and 14 days after (D14) infection; number indicates p-value of a Student’s t-test. Error bars indicate mean with ± SD. d) WT and KO mice were challenged with the model antigen 4-hydroxy-3-nitrophenylacetyl conjugated to chicken gamma globulin (NP-CGG). NP-specific IgG1 and IgM titers were quantified by ELISA from the serum isolated at the indicated days following immune challenge. Error bars indicate mean ± standard error of mean (SEM).

    Journal: bioRxiv

    Article Title: Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation

    doi: 10.1101/826370

    Figure Lengend Snippet: DOT1L-deficient B cells fail to mount an efficient immune response but show signs of plasma-cell differentiation upon challenge. a) Representative flow cytometry plots of splenic GC B cells 14 days after LCMV Armstrong infection in WT and Dot1L -KO mice and their statistical analyses. Error bars indicate mean with ± SD. b) Representative flow cytometry plots of plasma cells B from the spleen at 14 days after LCMV infection and their statistical analyses. Error bars indicate mean with ± SD. c) LCMV-specific IgM and IgG titers in the serum of WT and KO mice before (D0) and 14 days after (D14) infection; number indicates p-value of a Student’s t-test. Error bars indicate mean with ± SD. d) WT and KO mice were challenged with the model antigen 4-hydroxy-3-nitrophenylacetyl conjugated to chicken gamma globulin (NP-CGG). NP-specific IgG1 and IgM titers were quantified by ELISA from the serum isolated at the indicated days following immune challenge. Error bars indicate mean ± standard error of mean (SEM).

    Article Snippet: Next, HRP-conjugated IgG and IgM antibodies (Southern Biotech) were diluted 1:4000 in PBS/1% milk powder and incubated 1h at 37°C.

    Techniques: Cell Differentiation, Flow Cytometry, Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Isolation

    Validation of selected candidate biomarkers for HCC development in a larger patient population. (A) Quantification of plasma concentration of CPN (pg/mg plasma protein), CP, IgM, PON1 (μg/mg plasma protein), C4a (ng/mg plasma protein) and FGA (μg/mg plasma protein) by ELISA. The sample size was 24 in the control group and 22 in the tumor group. * Statistically significant differences between groups. Data as mean ± standard error; (B) ROC curves showing that the best predictive ability to identify patients with HCC belongs to the combination of C4a, FGA, CP and PON1.

    Journal: PLoS ONE

    Article Title: Plasma Protein Biomarkers of Hepatocellular Carcinoma in HCV-Infected Alcoholic Patients with Cirrhosis

    doi: 10.1371/journal.pone.0118527

    Figure Lengend Snippet: Validation of selected candidate biomarkers for HCC development in a larger patient population. (A) Quantification of plasma concentration of CPN (pg/mg plasma protein), CP, IgM, PON1 (μg/mg plasma protein), C4a (ng/mg plasma protein) and FGA (μg/mg plasma protein) by ELISA. The sample size was 24 in the control group and 22 in the tumor group. * Statistically significant differences between groups. Data as mean ± standard error; (B) ROC curves showing that the best predictive ability to identify patients with HCC belongs to the combination of C4a, FGA, CP and PON1.

    Article Snippet: Enzyme-linked immunosorbent assay (ELISA) ELISA tests were used to quantify the protein concentration of differentially expressed proteins CPN, CP, complement component 4a (C4a), fibrinogen-alpha (FGA), PON1 (USCN Life Science Inc., Wuhan, China), and immunoglobulin M (IgM) (Abcam, Cambridge, UK) in plasma samples from the control (24 cases) and tumor (22 cases) groups.

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

    Toso/FcμR binds IgM

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

    Article Title: Toso, a functional IgM receptor, is regulated by IL-2 in T and NK cells

    doi: 10.4049/jimmunol.1200840

    Figure Lengend Snippet: Toso/FcμR binds IgM

    Article Snippet: Flow cytometry verified the NK cells expressed Toso/FcμR and that SK-OV3 target cells could bind the IgM anti-Lewis Ab ( , bottom ).

    Techniques:

    GDC-0853 represses NF-κB–dependent gene transcription. CLL patient cells were treated with 1 µM BTK inhibitor for 72 hours with or without anti-IgM stimulation and assessed for gene expression changes of NF-κB targets by RT-PCR (N = 8). ** P ≤ .01; *** P ≤ .001.

    Journal: Blood

    Article Title: Noncovalent inhibition of C481S Bruton tyrosine kinase by GDC-0853: a new treatment strategy for ibrutinib-resistant CLL

    doi: 10.1182/blood-2017-10-809020

    Figure Lengend Snippet: GDC-0853 represses NF-κB–dependent gene transcription. CLL patient cells were treated with 1 µM BTK inhibitor for 72 hours with or without anti-IgM stimulation and assessed for gene expression changes of NF-κB targets by RT-PCR (N = 8). ** P ≤ .01; *** P ≤ .001.

    Article Snippet: CLL cells treated with GDC-0853 or ibrutinib were stimulated through their BCR by spinning onto a 6-well plate coated with 10 µg/mL anti-immunoglobulin M (anti-IgM) antibody (Jackson Laboratories).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Dynamics of humoral responses in mice after orally infected with T . gondii cysts. Detection of T . gondii -specific IgG (A) and IgM (B) in the serum of BALB/c mice (n = 3) infected with four cysts at 1, 3, 5, 7, 9, 11, 13, 15, 22, 30, 45 dpi with ELISA. Results are expressed as mean of the OD 450/630 ± S.D. Inverted black triangle p

    Journal: PLoS ONE

    Article Title: Interferon-Gamma Release Assay: An Effective Tool to Detect Early Toxoplasma gondii Infection in Mice

    doi: 10.1371/journal.pone.0137808

    Figure Lengend Snippet: Dynamics of humoral responses in mice after orally infected with T . gondii cysts. Detection of T . gondii -specific IgG (A) and IgM (B) in the serum of BALB/c mice (n = 3) infected with four cysts at 1, 3, 5, 7, 9, 11, 13, 15, 22, 30, 45 dpi with ELISA. Results are expressed as mean of the OD 450/630 ± S.D. Inverted black triangle p

    Article Snippet: Then 100 μl 1:2000 diluted HRP-labeled anti-mouse IgG (H + L) Ab (Proteintech Group, Inc., USA) or anti-mouse IgM Ab (Biosynthesis Biotech., China) was added to each well and the plates were incubated at 37°C for 1h followed by four time washes.

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