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  • 99
    Thermo Fisher igd
    Bmem markers on CNS infiltrating B cells have distinct surface expression from the periphery Brain and CLN cells isolated from pooled organs of JHMV infected mice at day 14 (CLN) and day 38 (brain) p.i. were analyzed for CD38 and <t>CD73</t> expression. (A) Representative gating strategy for CD19 + infiltrating B cells in the brain and gating of <t>IgD</t> + and IgM + subsets in the brain and CLN. Representative density plots depict CD38 (B–C) and CD73 (D–E) expression among total CD19 + , naïve IgD + IgM + , and IgD − IgM − isotype switched B cells within the CLN (B, D) and brain (C, E). Representative histograms depict CD38 or CD73 expression among naïve IgD + IgM + , activated IgD int IgM + , IgD − IgM + , and isotype-switched ASC/Bmem IgD − IgM − B cells within the CLN and brain. Data is representative of 3–4 independent experiments.
    Igd, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1014 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson immunoglobulin d igd fitc
    Bmem markers on CNS infiltrating B cells have distinct surface expression from the periphery Brain and CLN cells isolated from pooled organs of JHMV infected mice at day 14 (CLN) and day 38 (brain) p.i. were analyzed for CD38 and <t>CD73</t> expression. (A) Representative gating strategy for CD19 + infiltrating B cells in the brain and gating of <t>IgD</t> + and IgM + subsets in the brain and CLN. Representative density plots depict CD38 (B–C) and CD73 (D–E) expression among total CD19 + , naïve IgD + IgM + , and IgD − IgM − isotype switched B cells within the CLN (B, D) and brain (C, E). Representative histograms depict CD38 or CD73 expression among naïve IgD + IgM + , activated IgD int IgM + , IgD − IgM + , and isotype-switched ASC/Bmem IgD − IgM − B cells within the CLN and brain. Data is representative of 3–4 independent experiments.
    Immunoglobulin D Igd Fitc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Pharmingen anti immunoglobulin d igd
    Defective spleen organization in TNF/LTΔ3 mice. Magnifications are given on left. (A) Splenic cryosections of 6- to 8-week-old mice immunized with SRBC were stained to detect the distribution of T cells (CD3, blue) and B cells <t>(IgD</t> [brown], IgM), FDC (CR1), or stromal elements (ER-TR7) in TNF/LTΔ3 and single-knockout mice. WT, wild type. (B) Side-by-side comparison with TNF/LTα −/− mice. Splenic cryosections of 6- to 8-week-old nonimmunized mice were stained with anti-CD3 (blue), anti-B220 (brown), or anti-ER-TR7.
    Anti Immunoglobulin D Igd, supplied by Pharmingen, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    BioLegend immunoglobulin d
    Function of cTFH subsets and relative distribution after HSCT. (A) IgM production by naive B cells <t>(IgD</t> + CD27 − ) after in vitro coculture with different subsets of cTFH (CXCR5 + ) and non-TFH (CXCR5 − ) cells. (B) IgG production by naive B cells
    Immunoglobulin D, supplied by BioLegend, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Becton Dickinson anti immunoglobulin d igd pe
    Phenotypes of CD40-B cells and imDCs. Surface expression of B-cell maturation markers <t>IgD</t> and IgM (top panel), MHC-I and MHC-II (middle panel), and costimulatory molecules CD80, CD83 and CD86 (bottom panel) in resting B cells, CD40-B and imDCs were examined.
    Anti Immunoglobulin D Igd Pe, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore igd
    (a) Analysis of CD21 expression on peripheral blood (PB) lymphocytes and tonsillar (Tonsil) cells gated for immunoglobulin (Ig)M, <t>IgD,</t> IgG or IgA positivity. Cells were first stained indirectly for CD21, then stained for one of the four immunoglobulin isotypes. The numbers given represent the percentage of CD21-positive cells of the population, gated on the basis of immunoglobulin isotype. The example shown is representative of three identical experiments. (b) Analysis of human leucocyte antigen (HLA)-DR expression on PB lymphocytes and tonsillar cells gated for <t>IgM,</t> IgD, IgG or IgA positivity. B cells were first stained for HLA-DR and subsequently for one of the four immunoglobulin isotypes. The numbers given represent the percentage of HLA-DR-positive cells of the population, gated on the basis of immunoglobulin isotype. The example shown is representative of three identical experiments.
    Igd, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher igd apc
    (a) Analysis of CD21 expression on peripheral blood (PB) lymphocytes and tonsillar (Tonsil) cells gated for immunoglobulin (Ig)M, <t>IgD,</t> IgG or IgA positivity. Cells were first stained indirectly for CD21, then stained for one of the four immunoglobulin isotypes. The numbers given represent the percentage of CD21-positive cells of the population, gated on the basis of immunoglobulin isotype. The example shown is representative of three identical experiments. (b) Analysis of human leucocyte antigen (HLA)-DR expression on PB lymphocytes and tonsillar cells gated for <t>IgM,</t> IgD, IgG or IgA positivity. B cells were first stained for HLA-DR and subsequently for one of the four immunoglobulin isotypes. The numbers given represent the percentage of HLA-DR-positive cells of the population, gated on the basis of immunoglobulin isotype. The example shown is representative of three identical experiments.
    Igd Apc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Agilent technologies igd pe
    Positive association between PDC/MDC2 cell and isotype-switched memory <t>(IgD</t> − , CD27 + , <t>CD19</t> + ) B cell numbers in children with SPAD ( R -square=0.2102, p
    Igd Pe, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher igd pe
    Nfkb1 SSAA mutation impairs plasma cell differentiation. (A) Purified splenic FM B cells were cultured (triplicates) with α-IgM for 1 h or with LPS for 3 h. Expression of Irf4 and Prdm-1 mRNAs was determined by quantitative RT-PCR. Data were normalized to Hprt1 mRNA levels and represent mean fold change (±SEM) relative to unstimulated WT cells. All results are representative of at least three independent experiments (3 mice/genotype each). (B) Histograms show intracellular IRF4 staining in live purified FM B cells cultured ± LPS and IL-4 (40 h). (C) FM B cells were stimulated with LPS and IL-4 for 4 d. CD138 + <t>B220</t> lo plasma cells among the live (7AAD − ) lymphocytes were quantified by flow cytometry. (middle) Mean percentages of plasma cells (±SEM). (right) IgM secretion as quantified by ELISA (mean ± SEM). (D) Purified FM B cells were stimulated with LPS and IL-4 for 3 d. Fractions of IgG1 + live B cells (B220 + 7AAD − ) were determined by flow cytometry (±SEM; E) Graphs show the mean percentages (±SEM) of transferred CD45.2 + cells up-regulating intracellular IRF4 expression in the spleens of CD45.1 + WT hosts after 6d immunization with SRBC-HEL or unconjugated SRBC. These results are representative of four independent experiments ( n = 6 mice per condition). The gating strategies used are shown in Fig. S3 A . (F) Mean fluorescent intensities (MFI) of intracellular Bcl6 in transferred CD45.2 + B cells after 6-d immunization with SRBC-HEL or unconjugated SRBC. Results are representative of three independent experiments ( n = 6 mice per condition). Gating strategies used are shown in Fig. S4 B . (G) FM B cells were transduced with recombinant retroviruses. The fraction of GFP + cells differentiating to plasma cells (B220 lo <t>IgD</t> − CD138 + ) after stimulation with LPS and IL-4 was quantified by flow cytometry (±SEM). Results are representative of three independent experiments (3 mice/genotype each). (H) FM B cells were transduced with the indicated retroviruses (E.V., empty vector). The fraction of transferred GFP + CD45.2 + cells differentiating to GC B cells (B220 + PNA hi GL7 + CD45.2 + ) after 6-d immunization with SRBC-HEL or unconjugated SRBC are shown. Results are representative of two independent experiments ( n = 4–6 mice per condition). The gating strategies used are shown in Fig. S5. *, P
    Igd Pe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 488 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson pe igd
    Expression pattern of DNMTs in tonsillar B-cell subsets. ( A ) Cartoon illustrating the stages of B-cell development and T-dependent B-cell immune response. B cells are differentiated from hematopoietic stem cells (HSCs) in the bone marrow and released into the periphery upon maturation. Naive B cells encounter antigen-primed T cells in secondary lymphoid organs and enter the germinal center (GC) reaction followed by differentiation into memory B cells or antibody-secreting plasma cells (PCs). ( B ) Purification of B-cell subsets. CD20, <t>IgD,</t> and <t>CD38</t> cell surface expression were used to distinguish the cell populations: Naive B cells (CD20 + IgD + CD38 lo ), GC B cells (CD20 + IgD − CD38 + ), memory B cells (CD20 + IgD - CD38 − ), and PCs (CD20 lo/+ IgD − CD38 hi ). The percentage of each subset within tonsillar B cells is shown in the FACS plot in the middle panel. Naive, GC, memory, and PC populations were purified from eight individuals for gene expression and DNA methylation profiling on microarrays. The purity of each cell population ( > 90%) was confirmed by flow cytometry analysis of post-sorted cells ( right panel). ( C ) Transcript abundance of DNMT s in B-cell subsets by quantitative RT-PCR. DNMT expression levels are normalized to actin expression level in each cell type. The expression level in one biological replicate of naive B cells is arbitrarily set to one. Error bars denote standard deviation of three biological replicates. ( D ) Expression patterns of DNMT1 and DNMT3A in tonsils. ( Upper left , upper right ) Immunohistochemical staining of DNMT1 at 200× and 400× magnifications, respectively. ( Lower left , lower right ) Immunohistochemical staining of DNMT3A at 200× and 400× magnifications, respectively. ( E ). Scale bar, 50 μm.
    Pe Igd, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    igd  (Abcam)
    84
    Abcam igd
    Expression pattern of DNMTs in tonsillar B-cell subsets. ( A ) Cartoon illustrating the stages of B-cell development and T-dependent B-cell immune response. B cells are differentiated from hematopoietic stem cells (HSCs) in the bone marrow and released into the periphery upon maturation. Naive B cells encounter antigen-primed T cells in secondary lymphoid organs and enter the germinal center (GC) reaction followed by differentiation into memory B cells or antibody-secreting plasma cells (PCs). ( B ) Purification of B-cell subsets. CD20, <t>IgD,</t> and <t>CD38</t> cell surface expression were used to distinguish the cell populations: Naive B cells (CD20 + IgD + CD38 lo ), GC B cells (CD20 + IgD − CD38 + ), memory B cells (CD20 + IgD - CD38 − ), and PCs (CD20 lo/+ IgD − CD38 hi ). The percentage of each subset within tonsillar B cells is shown in the FACS plot in the middle panel. Naive, GC, memory, and PC populations were purified from eight individuals for gene expression and DNA methylation profiling on microarrays. The purity of each cell population ( > 90%) was confirmed by flow cytometry analysis of post-sorted cells ( right panel). ( C ) Transcript abundance of DNMT s in B-cell subsets by quantitative RT-PCR. DNMT expression levels are normalized to actin expression level in each cell type. The expression level in one biological replicate of naive B cells is arbitrarily set to one. Error bars denote standard deviation of three biological replicates. ( D ) Expression patterns of DNMT1 and DNMT3A in tonsils. ( Upper left , upper right ) Immunohistochemical staining of DNMT1 at 200× and 400× magnifications, respectively. ( Lower left , lower right ) Immunohistochemical staining of DNMT3A at 200× and 400× magnifications, respectively. ( E ). Scale bar, 50 μm.
    Igd, supplied by Abcam, used in various techniques. Bioz Stars score: 84/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioLegend igd pe
    Characterization of T cell receptor and immunoglobulin (Ig) expression on CD32 high <t>CD3</t> + CD4 + T cells. (A) Expression of TCRαβ on CD32-expressing CD3 + CD4 + T cell populations was measured by flow cytometry and is shown here as median fluorescence intensity. Bar is shown at the mean. (B) Histogram showing TCRαβ staining on CD32-expressing CD3 + CD4 + T cell populations of one sample shown in panel (A) . Fluorescence minus one (FMO) controls are shown as dashed lines and individual populations as solid lines. (C) Percentage expression of Ig was measured on CD3 + CD4 + CD32 high CD20 + cells. Expression of <t>IgD,</t> IgG, and IgM is shown. Expression was measured by flow cytometry, and bar is shown at the mean. (D) Representative staining of data shown in panel (C) .
    Igd Pe, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson igd apc
    Characterization of T cell receptor and immunoglobulin (Ig) expression on CD32 high <t>CD3</t> + CD4 + T cells. (A) Expression of TCRαβ on CD32-expressing CD3 + CD4 + T cell populations was measured by flow cytometry and is shown here as median fluorescence intensity. Bar is shown at the mean. (B) Histogram showing TCRαβ staining on CD32-expressing CD3 + CD4 + T cell populations of one sample shown in panel (A) . Fluorescence minus one (FMO) controls are shown as dashed lines and individual populations as solid lines. (C) Percentage expression of Ig was measured on CD3 + CD4 + CD32 high CD20 + cells. Expression of <t>IgD,</t> IgG, and IgM is shown. Expression was measured by flow cytometry, and bar is shown at the mean. (D) Representative staining of data shown in panel (C) .
    Igd Apc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson igd pecy7
    Characterization of T cell receptor and immunoglobulin (Ig) expression on CD32 high <t>CD3</t> + CD4 + T cells. (A) Expression of TCRαβ on CD32-expressing CD3 + CD4 + T cell populations was measured by flow cytometry and is shown here as median fluorescence intensity. Bar is shown at the mean. (B) Histogram showing TCRαβ staining on CD32-expressing CD3 + CD4 + T cell populations of one sample shown in panel (A) . Fluorescence minus one (FMO) controls are shown as dashed lines and individual populations as solid lines. (C) Percentage expression of Ig was measured on CD3 + CD4 + CD32 high CD20 + cells. Expression of <t>IgD,</t> IgG, and IgM is shown. Expression was measured by flow cytometry, and bar is shown at the mean. (D) Representative staining of data shown in panel (C) .
    Igd Pecy7, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    BioLegend igd af488
    Characterization of T cell receptor and immunoglobulin (Ig) expression on CD32 high <t>CD3</t> + CD4 + T cells. (A) Expression of TCRαβ on CD32-expressing CD3 + CD4 + T cell populations was measured by flow cytometry and is shown here as median fluorescence intensity. Bar is shown at the mean. (B) Histogram showing TCRαβ staining on CD32-expressing CD3 + CD4 + T cell populations of one sample shown in panel (A) . Fluorescence minus one (FMO) controls are shown as dashed lines and individual populations as solid lines. (C) Percentage expression of Ig was measured on CD3 + CD4 + CD32 high CD20 + cells. Expression of <t>IgD,</t> IgG, and IgM is shown. Expression was measured by flow cytometry, and bar is shown at the mean. (D) Representative staining of data shown in panel (C) .
    Igd Af488, supplied by BioLegend, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    BioLegend igd af700
    Characterization of T cell receptor and immunoglobulin (Ig) expression on CD32 high <t>CD3</t> + CD4 + T cells. (A) Expression of TCRαβ on CD32-expressing CD3 + CD4 + T cell populations was measured by flow cytometry and is shown here as median fluorescence intensity. Bar is shown at the mean. (B) Histogram showing TCRαβ staining on CD32-expressing CD3 + CD4 + T cell populations of one sample shown in panel (A) . Fluorescence minus one (FMO) controls are shown as dashed lines and individual populations as solid lines. (C) Percentage expression of Ig was measured on CD3 + CD4 + CD32 high CD20 + cells. Expression of <t>IgD,</t> IgG, and IgM is shown. Expression was measured by flow cytometry, and bar is shown at the mean. (D) Representative staining of data shown in panel (C) .
    Igd Af700, supplied by BioLegend, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    SouthernBiotech igd abs
    B cell depletion by CD20 and CD22 <t>mAbs</t> in NZB/W F 1 and C57BL/6 mice. CD20 ( A ), CD22 ( B ), or IgG2a control mAb (100 μg) was given i.p. to 8–10 wk-old mice with B cell numbers quantified on day 7. B220 + blood, B220 + spleen, B220 + peripheral lymph node, mature recirculating bone marrow <t>(IgD</t> + B220 hi ), marginal zone (B220 + CD21 hi CD1d hi ), and peritoneal B1 (B220 + IgM + CD11b + ) and B2 (B220 + IgM + CD11b − ) cell numbers were determined by immunofluorescence staining with flow cytometric analysis. Representative dot plots are shown for mAb-treated NZB/W F 1 mice with gated B cell percentages indicated. Significant differences between mean (±SEM) values for control mAb-treated (open bars) and CD20 or CD22 mAb-treated (filled bars) mice are indicated; *p
    Igd Abs, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    SouthernBiotech igd pe
    Phenotypic characterization of B cells from SW HEL mice. (A) Spleen cells from 10–14-wk-old NB6, LC2, V H 10 tar +/− , SW HEL (V H 10 tar +/− × LC2), and MD4 Ig Tg mice were harvested and stained with anti–B220-APC, HEL plus anti–HEL-FITC, <t>anti–IgD-PE,</t> and <t>anti–IgM-biotin</t> plus SA-PerCP. Cells were analyzed by flow cytometry and the frequency of HEL + (top box) and HEL − (bottom box) B cells in each density plot was indicated. Values from each mouse are representative of more than five independent experiments. (B) Spleen cells were stained as for A. IgM and IgD expression data are displayed for all B220 + B cells (NB6 and MD4, top) or specifically for HEL + and HEL − B cells (SW HEL , bottom) using the gates shown in A. Numbers indicate the proportions of gated cells within the indicated quadrants. Data are representative of more than five independent experiments. (C) Cells were isolated by peritoneal lavage from the indicated mice and stained with anti–B220-APC, HEL plus anti–HEL-FITC, anti–IgM-PE, and anti–CD5-biotin plus SA-PerCP. Cells were analyzed by flow cytometry and gated on live cells that were B220 + and/or IgM + to include both the B1 and B2 cell populations. For SW HEL mice the peritoneal cavity B cells were further gated into HEL + and HEL − subpopulations. Numbers indicate the proportion of gated cells in the B220 hi CD5 − window. Data are representative of three independent experiments.
    Igd Pe, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 91/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher igd efluor450
    Phenotypic characterization of B cells from SW HEL mice. (A) Spleen cells from 10–14-wk-old NB6, LC2, V H 10 tar +/− , SW HEL (V H 10 tar +/− × LC2), and MD4 Ig Tg mice were harvested and stained with anti–B220-APC, HEL plus anti–HEL-FITC, <t>anti–IgD-PE,</t> and <t>anti–IgM-biotin</t> plus SA-PerCP. Cells were analyzed by flow cytometry and the frequency of HEL + (top box) and HEL − (bottom box) B cells in each density plot was indicated. Values from each mouse are representative of more than five independent experiments. (B) Spleen cells were stained as for A. IgM and IgD expression data are displayed for all B220 + B cells (NB6 and MD4, top) or specifically for HEL + and HEL − B cells (SW HEL , bottom) using the gates shown in A. Numbers indicate the proportions of gated cells within the indicated quadrants. Data are representative of more than five independent experiments. (C) Cells were isolated by peritoneal lavage from the indicated mice and stained with anti–B220-APC, HEL plus anti–HEL-FITC, anti–IgM-PE, and anti–CD5-biotin plus SA-PerCP. Cells were analyzed by flow cytometry and gated on live cells that were B220 + and/or IgM + to include both the B1 and B2 cell populations. For SW HEL mice the peritoneal cavity B cells were further gated into HEL + and HEL − subpopulations. Numbers indicate the proportion of gated cells in the B220 hi CD5 − window. Data are representative of three independent experiments.
    Igd Efluor450, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Becton Dickinson igd h7
    Phenotypic characterization of B cells from SW HEL mice. (A) Spleen cells from 10–14-wk-old NB6, LC2, V H 10 tar +/− , SW HEL (V H 10 tar +/− × LC2), and MD4 Ig Tg mice were harvested and stained with anti–B220-APC, HEL plus anti–HEL-FITC, <t>anti–IgD-PE,</t> and <t>anti–IgM-biotin</t> plus SA-PerCP. Cells were analyzed by flow cytometry and the frequency of HEL + (top box) and HEL − (bottom box) B cells in each density plot was indicated. Values from each mouse are representative of more than five independent experiments. (B) Spleen cells were stained as for A. IgM and IgD expression data are displayed for all B220 + B cells (NB6 and MD4, top) or specifically for HEL + and HEL − B cells (SW HEL , bottom) using the gates shown in A. Numbers indicate the proportions of gated cells within the indicated quadrants. Data are representative of more than five independent experiments. (C) Cells were isolated by peritoneal lavage from the indicated mice and stained with anti–B220-APC, HEL plus anti–HEL-FITC, anti–IgM-PE, and anti–CD5-biotin plus SA-PerCP. Cells were analyzed by flow cytometry and gated on live cells that were B220 + and/or IgM + to include both the B1 and B2 cell populations. For SW HEL mice the peritoneal cavity B cells were further gated into HEL + and HEL − subpopulations. Numbers indicate the proportion of gated cells in the B220 hi CD5 − window. Data are representative of three independent experiments.
    Igd H7, supplied by Becton Dickinson, 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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    86
    Thermo Fisher igd vioblue
    Phenotypic characterization of B cells from SW HEL mice. (A) Spleen cells from 10–14-wk-old NB6, LC2, V H 10 tar +/− , SW HEL (V H 10 tar +/− × LC2), and MD4 Ig Tg mice were harvested and stained with anti–B220-APC, HEL plus anti–HEL-FITC, <t>anti–IgD-PE,</t> and <t>anti–IgM-biotin</t> plus SA-PerCP. Cells were analyzed by flow cytometry and the frequency of HEL + (top box) and HEL − (bottom box) B cells in each density plot was indicated. Values from each mouse are representative of more than five independent experiments. (B) Spleen cells were stained as for A. IgM and IgD expression data are displayed for all B220 + B cells (NB6 and MD4, top) or specifically for HEL + and HEL − B cells (SW HEL , bottom) using the gates shown in A. Numbers indicate the proportions of gated cells within the indicated quadrants. Data are representative of more than five independent experiments. (C) Cells were isolated by peritoneal lavage from the indicated mice and stained with anti–B220-APC, HEL plus anti–HEL-FITC, anti–IgM-PE, and anti–CD5-biotin plus SA-PerCP. Cells were analyzed by flow cytometry and gated on live cells that were B220 + and/or IgM + to include both the B1 and B2 cell populations. For SW HEL mice the peritoneal cavity B cells were further gated into HEL + and HEL − subpopulations. Numbers indicate the proportion of gated cells in the B220 hi CD5 − window. Data are representative of three independent experiments.
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    Image Search Results


    Bmem markers on CNS infiltrating B cells have distinct surface expression from the periphery Brain and CLN cells isolated from pooled organs of JHMV infected mice at day 14 (CLN) and day 38 (brain) p.i. were analyzed for CD38 and CD73 expression. (A) Representative gating strategy for CD19 + infiltrating B cells in the brain and gating of IgD + and IgM + subsets in the brain and CLN. Representative density plots depict CD38 (B–C) and CD73 (D–E) expression among total CD19 + , naïve IgD + IgM + , and IgD − IgM − isotype switched B cells within the CLN (B, D) and brain (C, E). Representative histograms depict CD38 or CD73 expression among naïve IgD + IgM + , activated IgD int IgM + , IgD − IgM + , and isotype-switched ASC/Bmem IgD − IgM − B cells within the CLN and brain. Data is representative of 3–4 independent experiments.

    Journal: Journal of neuroscience methods

    Article Title: An optimized method for enumerating CNS derived memory B cells during viral-induced inflammation

    doi: 10.1016/j.jneumeth.2017.05.011

    Figure Lengend Snippet: Bmem markers on CNS infiltrating B cells have distinct surface expression from the periphery Brain and CLN cells isolated from pooled organs of JHMV infected mice at day 14 (CLN) and day 38 (brain) p.i. were analyzed for CD38 and CD73 expression. (A) Representative gating strategy for CD19 + infiltrating B cells in the brain and gating of IgD + and IgM + subsets in the brain and CLN. Representative density plots depict CD38 (B–C) and CD73 (D–E) expression among total CD19 + , naïve IgD + IgM + , and IgD − IgM − isotype switched B cells within the CLN (B, D) and brain (C, E). Representative histograms depict CD38 or CD73 expression among naïve IgD + IgM + , activated IgD int IgM + , IgD − IgM + , and isotype-switched ASC/Bmem IgD − IgM − B cells within the CLN and brain. Data is representative of 3–4 independent experiments.

    Article Snippet: Expression of cell surface markers was determined by staining on ice for 30 minutes with Ab specific for CD45 (30-F11; PerCP-Cy5.5), CD19 (1D3; PE-CF594), CD73 (AD2: PE-Cy7) (BD Biosciences, San Jose, CA), IgM (eB131-15F9; PE), IgD (11–26; APC), and CD38 (90; PE) (eBioscience, San Diego, CA).

    Techniques: Expressing, Isolation, Infection, Mouse Assay

    Defective spleen organization in TNF/LTΔ3 mice. Magnifications are given on left. (A) Splenic cryosections of 6- to 8-week-old mice immunized with SRBC were stained to detect the distribution of T cells (CD3, blue) and B cells (IgD [brown], IgM), FDC (CR1), or stromal elements (ER-TR7) in TNF/LTΔ3 and single-knockout mice. WT, wild type. (B) Side-by-side comparison with TNF/LTα −/− mice. Splenic cryosections of 6- to 8-week-old nonimmunized mice were stained with anti-CD3 (blue), anti-B220 (brown), or anti-ER-TR7.

    Journal: Molecular and Cellular Biology

    Article Title: Redundancy in Tumor Necrosis Factor (TNF) and Lymphotoxin (LT) Signaling In Vivo: Mice with Inactivation of the Entire TNF/LT Locus versus Single-Knockout Mice

    doi: 10.1128/MCB.22.24.8626-8634.2002

    Figure Lengend Snippet: Defective spleen organization in TNF/LTΔ3 mice. Magnifications are given on left. (A) Splenic cryosections of 6- to 8-week-old mice immunized with SRBC were stained to detect the distribution of T cells (CD3, blue) and B cells (IgD [brown], IgM), FDC (CR1), or stromal elements (ER-TR7) in TNF/LTΔ3 and single-knockout mice. WT, wild type. (B) Side-by-side comparison with TNF/LTα −/− mice. Splenic cryosections of 6- to 8-week-old nonimmunized mice were stained with anti-CD3 (blue), anti-B220 (brown), or anti-ER-TR7.

    Article Snippet: The following rat anti-mouse monoclonal antibodies were used: anti-CD3, anti-immunoglobulin D (IgD), anti-B220 (PharMingen), ER-TR7 (Biogenesis, Poole, United Kingdom), MOMA1 (Research Diagnostics, Flanders, N.J.), and FDC-M1 (generously provided by M. Kosco-Vilbois [Serono Pharmaceutical Research Institute, Geneva, Switzerland]).

    Techniques: Mouse Assay, Staining, Knock-Out

    Function of cTFH subsets and relative distribution after HSCT. (A) IgM production by naive B cells (IgD + CD27 − ) after in vitro coculture with different subsets of cTFH (CXCR5 + ) and non-TFH (CXCR5 − ) cells. (B) IgG production by naive B cells

    Journal: Blood

    Article Title: Circulating T follicular helper cells with increased function during chronic graft-versus-host disease

    doi: 10.1182/blood-2015-12-688895

    Figure Lengend Snippet: Function of cTFH subsets and relative distribution after HSCT. (A) IgM production by naive B cells (IgD + CD27 − ) after in vitro coculture with different subsets of cTFH (CXCR5 + ) and non-TFH (CXCR5 − ) cells. (B) IgG production by naive B cells

    Article Snippet: The following reagents were used: CD3 (clone UCHT1), CD4 (SK3), CXCR5 (RF8B2), CXCR3 (1C6/CXCR3), CCR6 (11A9), CD38 (HIT2) from BD Biosciences; CD45RA (IMU2711) from Beckman; ICOS (C398.4A), PD-1 (EH12.2H7), CD19 (HIB19), immunoglobulin D (IgD; IA6-2) from BioLegend; and CD27 (O323) from eBioscience.

    Techniques: In Vitro

    Phenotypes of CD40-B cells and imDCs. Surface expression of B-cell maturation markers IgD and IgM (top panel), MHC-I and MHC-II (middle panel), and costimulatory molecules CD80, CD83 and CD86 (bottom panel) in resting B cells, CD40-B and imDCs were examined.

    Journal: Cellular and Molecular Immunology

    Article Title: CD40-activated B cells are more potent than immature dendritic cells to induce and expand CD4+ regulatory T cells

    doi: 10.1038/cmi.2009.103

    Figure Lengend Snippet: Phenotypes of CD40-B cells and imDCs. Surface expression of B-cell maturation markers IgD and IgM (top panel), MHC-I and MHC-II (middle panel), and costimulatory molecules CD80, CD83 and CD86 (bottom panel) in resting B cells, CD40-B and imDCs were examined.

    Article Snippet: The following fluorescence-conjugated mAbs were used: anti-CD4-Alexa-405 (Caltag, Burlingame, CA, USA), anti-CD25-allophycocyanin, anti-CD3-FITC, anti-CD19-allophycocyanin, anti-major histocompability complex (MHC)-I-FITC, anti-MHC-II-FITC, anti-CD80-FITC, anti-CD83-FITC, anti-CD86-FITC, anti-immunoglobulin M (IgM)-phycoerythrin (PE)-Cy-5, anti-immunoglobulin D (IgD)-PE, anti-cytotoxic T-lymphocyte-associated antigen (CTLA)-4-PE, anti-glucocorticoid-induced tumor necrosis factor receptor-PE, anti-CD103; these fluorescent-conjugated mAbs and isotype-matched control Abs of irrelevant specificity were purchased from BD Biosciences (San Jose, CA, USA).

    Techniques: Expressing

    (a) Analysis of CD21 expression on peripheral blood (PB) lymphocytes and tonsillar (Tonsil) cells gated for immunoglobulin (Ig)M, IgD, IgG or IgA positivity. Cells were first stained indirectly for CD21, then stained for one of the four immunoglobulin isotypes. The numbers given represent the percentage of CD21-positive cells of the population, gated on the basis of immunoglobulin isotype. The example shown is representative of three identical experiments. (b) Analysis of human leucocyte antigen (HLA)-DR expression on PB lymphocytes and tonsillar cells gated for IgM, IgD, IgG or IgA positivity. B cells were first stained for HLA-DR and subsequently for one of the four immunoglobulin isotypes. The numbers given represent the percentage of HLA-DR-positive cells of the population, gated on the basis of immunoglobulin isotype. The example shown is representative of three identical experiments.

    Journal: Immunology

    Article Title: B-lymphocyte subpopulations are equally susceptible to Epstein-Barr virus infection, irrespective of immunoglobulin isotype expression

    doi: 10.1046/j.1365-2567.2003.01601.x

    Figure Lengend Snippet: (a) Analysis of CD21 expression on peripheral blood (PB) lymphocytes and tonsillar (Tonsil) cells gated for immunoglobulin (Ig)M, IgD, IgG or IgA positivity. Cells were first stained indirectly for CD21, then stained for one of the four immunoglobulin isotypes. The numbers given represent the percentage of CD21-positive cells of the population, gated on the basis of immunoglobulin isotype. The example shown is representative of three identical experiments. (b) Analysis of human leucocyte antigen (HLA)-DR expression on PB lymphocytes and tonsillar cells gated for IgM, IgD, IgG or IgA positivity. B cells were first stained for HLA-DR and subsequently for one of the four immunoglobulin isotypes. The numbers given represent the percentage of HLA-DR-positive cells of the population, gated on the basis of immunoglobulin isotype. The example shown is representative of three identical experiments.

    Article Snippet: Then, after three washes in PBS to remove the conjugate, FITC–rabbit antibody to human IgM, IgD, IgG, or IgA was added for 45 min, after which the cytospins were washed three times before mounting with 80% glycerol solution in PBS containing 2·5% 1,4-diazabicyclo-(2.2.2) octane (Sigma).

    Techniques: Expressing, Staining

    Positive association between PDC/MDC2 cell and isotype-switched memory (IgD − , CD27 + , CD19 + ) B cell numbers in children with SPAD ( R -square=0.2102, p

    Journal: European journal of pediatrics

    Article Title: Age-dependent changes in peripheral blood dendritic cell subsets in normal children and children with specific polysaccharide antibody deficiency (SPAD)

    doi: 10.1007/s00431-010-1210-y

    Figure Lengend Snippet: Positive association between PDC/MDC2 cell and isotype-switched memory (IgD − , CD27 + , CD19 + ) B cell numbers in children with SPAD ( R -square=0.2102, p

    Article Snippet: Memory B cells (IgD− , CD27+ , and CD19+ B cells) were detected by staining with anti-CD45-PITC, anti-CD19-APC-Cy7, CD27-APC (all from BD biosciences, San Jose CA), and IgD-PE (DAKO, Carpinteria, CA) monoclonal antibodies [ ].

    Techniques:

    Nfkb1 SSAA mutation impairs plasma cell differentiation. (A) Purified splenic FM B cells were cultured (triplicates) with α-IgM for 1 h or with LPS for 3 h. Expression of Irf4 and Prdm-1 mRNAs was determined by quantitative RT-PCR. Data were normalized to Hprt1 mRNA levels and represent mean fold change (±SEM) relative to unstimulated WT cells. All results are representative of at least three independent experiments (3 mice/genotype each). (B) Histograms show intracellular IRF4 staining in live purified FM B cells cultured ± LPS and IL-4 (40 h). (C) FM B cells were stimulated with LPS and IL-4 for 4 d. CD138 + B220 lo plasma cells among the live (7AAD − ) lymphocytes were quantified by flow cytometry. (middle) Mean percentages of plasma cells (±SEM). (right) IgM secretion as quantified by ELISA (mean ± SEM). (D) Purified FM B cells were stimulated with LPS and IL-4 for 3 d. Fractions of IgG1 + live B cells (B220 + 7AAD − ) were determined by flow cytometry (±SEM; E) Graphs show the mean percentages (±SEM) of transferred CD45.2 + cells up-regulating intracellular IRF4 expression in the spleens of CD45.1 + WT hosts after 6d immunization with SRBC-HEL or unconjugated SRBC. These results are representative of four independent experiments ( n = 6 mice per condition). The gating strategies used are shown in Fig. S3 A . (F) Mean fluorescent intensities (MFI) of intracellular Bcl6 in transferred CD45.2 + B cells after 6-d immunization with SRBC-HEL or unconjugated SRBC. Results are representative of three independent experiments ( n = 6 mice per condition). Gating strategies used are shown in Fig. S4 B . (G) FM B cells were transduced with recombinant retroviruses. The fraction of GFP + cells differentiating to plasma cells (B220 lo IgD − CD138 + ) after stimulation with LPS and IL-4 was quantified by flow cytometry (±SEM). Results are representative of three independent experiments (3 mice/genotype each). (H) FM B cells were transduced with the indicated retroviruses (E.V., empty vector). The fraction of transferred GFP + CD45.2 + cells differentiating to GC B cells (B220 + PNA hi GL7 + CD45.2 + ) after 6-d immunization with SRBC-HEL or unconjugated SRBC are shown. Results are representative of two independent experiments ( n = 4–6 mice per condition). The gating strategies used are shown in Fig. S5. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKK-induced NF-κB1 p105 proteolysis is critical for B cell antibody responses to T cell–dependent antigen

    doi: 10.1084/jem.20132019

    Figure Lengend Snippet: Nfkb1 SSAA mutation impairs plasma cell differentiation. (A) Purified splenic FM B cells were cultured (triplicates) with α-IgM for 1 h or with LPS for 3 h. Expression of Irf4 and Prdm-1 mRNAs was determined by quantitative RT-PCR. Data were normalized to Hprt1 mRNA levels and represent mean fold change (±SEM) relative to unstimulated WT cells. All results are representative of at least three independent experiments (3 mice/genotype each). (B) Histograms show intracellular IRF4 staining in live purified FM B cells cultured ± LPS and IL-4 (40 h). (C) FM B cells were stimulated with LPS and IL-4 for 4 d. CD138 + B220 lo plasma cells among the live (7AAD − ) lymphocytes were quantified by flow cytometry. (middle) Mean percentages of plasma cells (±SEM). (right) IgM secretion as quantified by ELISA (mean ± SEM). (D) Purified FM B cells were stimulated with LPS and IL-4 for 3 d. Fractions of IgG1 + live B cells (B220 + 7AAD − ) were determined by flow cytometry (±SEM; E) Graphs show the mean percentages (±SEM) of transferred CD45.2 + cells up-regulating intracellular IRF4 expression in the spleens of CD45.1 + WT hosts after 6d immunization with SRBC-HEL or unconjugated SRBC. These results are representative of four independent experiments ( n = 6 mice per condition). The gating strategies used are shown in Fig. S3 A . (F) Mean fluorescent intensities (MFI) of intracellular Bcl6 in transferred CD45.2 + B cells after 6-d immunization with SRBC-HEL or unconjugated SRBC. Results are representative of three independent experiments ( n = 6 mice per condition). Gating strategies used are shown in Fig. S4 B . (G) FM B cells were transduced with recombinant retroviruses. The fraction of GFP + cells differentiating to plasma cells (B220 lo IgD − CD138 + ) after stimulation with LPS and IL-4 was quantified by flow cytometry (±SEM). Results are representative of three independent experiments (3 mice/genotype each). (H) FM B cells were transduced with the indicated retroviruses (E.V., empty vector). The fraction of transferred GFP + CD45.2 + cells differentiating to GC B cells (B220 + PNA hi GL7 + CD45.2 + ) after 6-d immunization with SRBC-HEL or unconjugated SRBC are shown. Results are representative of two independent experiments ( n = 4–6 mice per condition). The gating strategies used are shown in Fig. S5. *, P

    Article Snippet: GC B cells were stained using GL7-FITC mAb (Ly77, BD), revealed with rabbit anti-FITC (Alexa Fluor 488 Signal-Amplification kit; Invitrogen), IgD-PE (11-26c; eBioscience), and B220-Alexa Fluor 647 (RA3-6B2, BD).

    Techniques: Mutagenesis, Cell Differentiation, Purification, Cell Culture, Expressing, Quantitative RT-PCR, Mouse Assay, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Transduction, Recombinant, Plasmid Preparation

    Nfkb1 SSAA/SSAA mutation blocks the B cell antibody response to a TD antigen. WT or Nfkb1 SSAA/SSAA BM cells were mixed with μMT −/− BM cells in a 1:4 ratio, and transferred into sublethally irradiated Rag1 −/− mice. After 8 wk, chimeras were immunized with NP 27 -CGG alum or PBS alum controls. (A) Serum antibody response to NP 27 -CGG immunization of WT or Nfkb1 SSAA/SSAA mixed BM chimeras assessed 7 and 14 d after challenge. Data show mean NP-specific serum IgM and IgG1 levels (±SEM) measured by ELISA. (B) Flow cytometric analysis of total naive B cells (IgM + B220 + ; left) and αNP-IgG1 + plasmablasts (PB; B220 lo IgM lo IgD − CD138 + ; right) in the spleens of chimeras 7 d after NP 27 -CGG immunization (mean absolute number ± SEM). (C) Flow cytometric analysis of total GC B cells (IgM lo B220 + IgD − PNA hi GL7 + ; left) and αNP-IgG1 + GC B cells (right) in chimeras 14 d after NP 27 -CGG immunization (mean absolute number ± SEM). (D) Confocal microscopy of spleen sections from WT or Nfkb1 SSAA/SSAA chimeras immunized with NP 27 -CGG (14d). GC formation was measured by staining with anti-IgD (red), anti-B220 (blue) and GL7 (green). In A–D, results are representative of at least three independent experiments with n = 5 mice per genotype. (E) WT or Nfkb1 SSAA/SSAA CD45.2 + BM cells were mixed with WT CD45.1 + and μMT −/− BM cells in a 1:1:3 ratio, and transferred into sublethally irradiated Rag1 −/− mice. After an 8-wk reconstitution, chimeras were immunized with NP 27 -CGG alum or alum alone. Graph shows the flow cytometric analysis of αNP + IgG1 + plasmablasts (PB), 7 and 14 d after NP 27 -CGG immunization. Results are representative of at least two independent experiments with n = 5–6 mice per genotype. **, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKK-induced NF-κB1 p105 proteolysis is critical for B cell antibody responses to T cell–dependent antigen

    doi: 10.1084/jem.20132019

    Figure Lengend Snippet: Nfkb1 SSAA/SSAA mutation blocks the B cell antibody response to a TD antigen. WT or Nfkb1 SSAA/SSAA BM cells were mixed with μMT −/− BM cells in a 1:4 ratio, and transferred into sublethally irradiated Rag1 −/− mice. After 8 wk, chimeras were immunized with NP 27 -CGG alum or PBS alum controls. (A) Serum antibody response to NP 27 -CGG immunization of WT or Nfkb1 SSAA/SSAA mixed BM chimeras assessed 7 and 14 d after challenge. Data show mean NP-specific serum IgM and IgG1 levels (±SEM) measured by ELISA. (B) Flow cytometric analysis of total naive B cells (IgM + B220 + ; left) and αNP-IgG1 + plasmablasts (PB; B220 lo IgM lo IgD − CD138 + ; right) in the spleens of chimeras 7 d after NP 27 -CGG immunization (mean absolute number ± SEM). (C) Flow cytometric analysis of total GC B cells (IgM lo B220 + IgD − PNA hi GL7 + ; left) and αNP-IgG1 + GC B cells (right) in chimeras 14 d after NP 27 -CGG immunization (mean absolute number ± SEM). (D) Confocal microscopy of spleen sections from WT or Nfkb1 SSAA/SSAA chimeras immunized with NP 27 -CGG (14d). GC formation was measured by staining with anti-IgD (red), anti-B220 (blue) and GL7 (green). In A–D, results are representative of at least three independent experiments with n = 5 mice per genotype. (E) WT or Nfkb1 SSAA/SSAA CD45.2 + BM cells were mixed with WT CD45.1 + and μMT −/− BM cells in a 1:1:3 ratio, and transferred into sublethally irradiated Rag1 −/− mice. After an 8-wk reconstitution, chimeras were immunized with NP 27 -CGG alum or alum alone. Graph shows the flow cytometric analysis of αNP + IgG1 + plasmablasts (PB), 7 and 14 d after NP 27 -CGG immunization. Results are representative of at least two independent experiments with n = 5–6 mice per genotype. **, P

    Article Snippet: GC B cells were stained using GL7-FITC mAb (Ly77, BD), revealed with rabbit anti-FITC (Alexa Fluor 488 Signal-Amplification kit; Invitrogen), IgD-PE (11-26c; eBioscience), and B220-Alexa Fluor 647 (RA3-6B2, BD).

    Techniques: Mutagenesis, Irradiation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Confocal Microscopy, Staining

    Nfkb1 SSAA/SSAA B cells migrate normally to follicle T cell zone. (A) Flow cytometric analysis of CCR7 surface expression on purified FM B cells, cultured ± α-IgM for 6 h. Graphs represent CCR7 mean fluorescent intensity (±SEM) of live B cells (7AAD − B220 + ). (B) Triplicate cultures of purified splenic FM B cells cultured ± α-IgM for 1 h. Expression of Ebi-2 mRNAs was determined by quantitative RT-PCR. Data were normalized to Hprt1 mRNA levels and represent mean fold change (±SEM) relative to unstimulated WT cells. All results are representative of at least two independent experiments done, with 3 mice/genotype each. (C) Splenic distribution of WT and Nfkb1 SSAA/SSAA CD45.2 + B cells, prestimulated in vitro for 1 h with α-IgM or control buffer (−), 5.5 h after transfer in to CD45.1 + WT hosts. Transferred B cells were detected by staining for CD45.2 (green), whereas host B cells and T cells were stained with antibodies to IgD (red) and CD4 (blue), respectively. Views are representative of at least two mice of each type. (D) Purified antigen-pulsed (Ag + ) or not pulsed (Ag − ) B cells were co-culture with purified CTV-labeled OTII CD4 + T cells for 72 h. The fraction of OTII CD4 + that had divided was determined by flow cytometric analysis of CTV dilution (mean ± SEM; triplicates). All results are representative of at least two independent experiments done, with 3 mice/genotype each.

    Journal: The Journal of Experimental Medicine

    Article Title: IKK-induced NF-κB1 p105 proteolysis is critical for B cell antibody responses to T cell–dependent antigen

    doi: 10.1084/jem.20132019

    Figure Lengend Snippet: Nfkb1 SSAA/SSAA B cells migrate normally to follicle T cell zone. (A) Flow cytometric analysis of CCR7 surface expression on purified FM B cells, cultured ± α-IgM for 6 h. Graphs represent CCR7 mean fluorescent intensity (±SEM) of live B cells (7AAD − B220 + ). (B) Triplicate cultures of purified splenic FM B cells cultured ± α-IgM for 1 h. Expression of Ebi-2 mRNAs was determined by quantitative RT-PCR. Data were normalized to Hprt1 mRNA levels and represent mean fold change (±SEM) relative to unstimulated WT cells. All results are representative of at least two independent experiments done, with 3 mice/genotype each. (C) Splenic distribution of WT and Nfkb1 SSAA/SSAA CD45.2 + B cells, prestimulated in vitro for 1 h with α-IgM or control buffer (−), 5.5 h after transfer in to CD45.1 + WT hosts. Transferred B cells were detected by staining for CD45.2 (green), whereas host B cells and T cells were stained with antibodies to IgD (red) and CD4 (blue), respectively. Views are representative of at least two mice of each type. (D) Purified antigen-pulsed (Ag + ) or not pulsed (Ag − ) B cells were co-culture with purified CTV-labeled OTII CD4 + T cells for 72 h. The fraction of OTII CD4 + that had divided was determined by flow cytometric analysis of CTV dilution (mean ± SEM; triplicates). All results are representative of at least two independent experiments done, with 3 mice/genotype each.

    Article Snippet: GC B cells were stained using GL7-FITC mAb (Ly77, BD), revealed with rabbit anti-FITC (Alexa Fluor 488 Signal-Amplification kit; Invitrogen), IgD-PE (11-26c; eBioscience), and B220-Alexa Fluor 647 (RA3-6B2, BD).

    Techniques: Flow Cytometry, Expressing, Purification, Cell Culture, Quantitative RT-PCR, Mouse Assay, In Vitro, Staining, Co-Culture Assay, Labeling

    TPL-2 is not required for a B cell antibody response to TD antigen. WT or Map3k8 −/− BM cells were mixed with μMT −/− BM cells in a 1:4 ratio, and transferred into sublethally irradiated Rag1 −/− mice. After 8 wk, chimeras were immunized with NP 27 -CGG alum or PBS alum controls. (A) Flow cytometric analysis of total naive B cells (IgM + B220 + ). (B) α-NP IgM and IgG1 serum antibody levels were quantified by ELISA (mean ± SEM), 14 d after NP 27 -CGG immunization. (C) Splenic α-NP IgG1 + plasmablasts (PB; B220 lo IgM lo IgD − CD138 + ) 7d after NP 27 -CGG immunization (mean absolute number ± SEM). (D) Splenic α-NP IgG1 + GC B cells 14 d after NP 27 -CGG immunization (mean absolute number ± SEM). All results are representative of two independent experiments (4–5 mice per genotype each).

    Journal: The Journal of Experimental Medicine

    Article Title: IKK-induced NF-κB1 p105 proteolysis is critical for B cell antibody responses to T cell–dependent antigen

    doi: 10.1084/jem.20132019

    Figure Lengend Snippet: TPL-2 is not required for a B cell antibody response to TD antigen. WT or Map3k8 −/− BM cells were mixed with μMT −/− BM cells in a 1:4 ratio, and transferred into sublethally irradiated Rag1 −/− mice. After 8 wk, chimeras were immunized with NP 27 -CGG alum or PBS alum controls. (A) Flow cytometric analysis of total naive B cells (IgM + B220 + ). (B) α-NP IgM and IgG1 serum antibody levels were quantified by ELISA (mean ± SEM), 14 d after NP 27 -CGG immunization. (C) Splenic α-NP IgG1 + plasmablasts (PB; B220 lo IgM lo IgD − CD138 + ) 7d after NP 27 -CGG immunization (mean absolute number ± SEM). (D) Splenic α-NP IgG1 + GC B cells 14 d after NP 27 -CGG immunization (mean absolute number ± SEM). All results are representative of two independent experiments (4–5 mice per genotype each).

    Article Snippet: GC B cells were stained using GL7-FITC mAb (Ly77, BD), revealed with rabbit anti-FITC (Alexa Fluor 488 Signal-Amplification kit; Invitrogen), IgD-PE (11-26c; eBioscience), and B220-Alexa Fluor 647 (RA3-6B2, BD).

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

    Nfkb1 SSAA/SSAA mice have normal numbers of FM B cells. (A–D) Flow cytometric analysis of B cell populations in WT and Nfkb1 SSAA/SSAA mice from the indicated organs. The gating strategies used are shown in Fig. S1 . (A) Absolute cell numbers of pro–B (B220 + CD19 + IgD − IgM − CD2 − ), pre–B (B220 + CD19 + IgD − IgM − CD2 + ), immature mature B (B220 + CD19 + IgD − IgM + CD2 + ), and mature B cells (B220 + CD19 + IgD + IgM + CD2 + ) in the BM (mean ± SEM; n = 10–11 mice/genotype). (B) Absolute cell numbers (mean ± SEM; n = 10–11 mice/genotype) of total B cells (IgM + IgD + ), immature B cells (IgD lo B220 + AA4.1 + ) separated into transitional T1 B cells (IgM hi CD23 − ), and T2 B cells (IgM hi CD23 + ), and mature B cells (IgD hi B220 + AA4.1 − ) separated into FM B cells (IgM hi CD23 + ) and MZ B cells (IgM hi CD23 − ) in the spleen. (C) Percentages of B cells (IgM + CD19 + ) in peripheral lymph nodes (pools of single cervical, axillary and inguinal nodes; mean ± SEM; n = 10–11 mice/genotype). (D) Proportion of B1a (B220 + CD19 + CD5 + CD23 − ), B1b (B220 + CD19 + CD5 − CD23 − ), and B2 (B220 + CD19 + CD5 − CD23 + ) cells in the peritoneal cavity (mean ± SEM; n = 5–7 mice/genotype). In A–D, results are representative of at least three independent experiments. (E) WT or Nfkb1 SSAA/SSAA CD45.2 + BM cells were mixed with WT CD45.1 + BM cells at a 1:1 ratio, and transferred into sublethally irradiated Rag1 −/− mice. After 8-wk reconstitution, the absolute number of CD45.2 + MZ B cells was assessed by flow cytometry. FM B cells were used as the control population. Graphs show absolute cell numbers (mean ± SEM; n = 14 mice/genotype), results are representative of two independent experiments. (F) Graphs show ELISAs for IgM, IgA, IgG1, and IgG2b, assaying sera from naive WT and Nfkb1 SSAA/SSAA mice. n = 18 mice per genotype. PEC, peritoneal cavity. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKK-induced NF-κB1 p105 proteolysis is critical for B cell antibody responses to T cell–dependent antigen

    doi: 10.1084/jem.20132019

    Figure Lengend Snippet: Nfkb1 SSAA/SSAA mice have normal numbers of FM B cells. (A–D) Flow cytometric analysis of B cell populations in WT and Nfkb1 SSAA/SSAA mice from the indicated organs. The gating strategies used are shown in Fig. S1 . (A) Absolute cell numbers of pro–B (B220 + CD19 + IgD − IgM − CD2 − ), pre–B (B220 + CD19 + IgD − IgM − CD2 + ), immature mature B (B220 + CD19 + IgD − IgM + CD2 + ), and mature B cells (B220 + CD19 + IgD + IgM + CD2 + ) in the BM (mean ± SEM; n = 10–11 mice/genotype). (B) Absolute cell numbers (mean ± SEM; n = 10–11 mice/genotype) of total B cells (IgM + IgD + ), immature B cells (IgD lo B220 + AA4.1 + ) separated into transitional T1 B cells (IgM hi CD23 − ), and T2 B cells (IgM hi CD23 + ), and mature B cells (IgD hi B220 + AA4.1 − ) separated into FM B cells (IgM hi CD23 + ) and MZ B cells (IgM hi CD23 − ) in the spleen. (C) Percentages of B cells (IgM + CD19 + ) in peripheral lymph nodes (pools of single cervical, axillary and inguinal nodes; mean ± SEM; n = 10–11 mice/genotype). (D) Proportion of B1a (B220 + CD19 + CD5 + CD23 − ), B1b (B220 + CD19 + CD5 − CD23 − ), and B2 (B220 + CD19 + CD5 − CD23 + ) cells in the peritoneal cavity (mean ± SEM; n = 5–7 mice/genotype). In A–D, results are representative of at least three independent experiments. (E) WT or Nfkb1 SSAA/SSAA CD45.2 + BM cells were mixed with WT CD45.1 + BM cells at a 1:1 ratio, and transferred into sublethally irradiated Rag1 −/− mice. After 8-wk reconstitution, the absolute number of CD45.2 + MZ B cells was assessed by flow cytometry. FM B cells were used as the control population. Graphs show absolute cell numbers (mean ± SEM; n = 14 mice/genotype), results are representative of two independent experiments. (F) Graphs show ELISAs for IgM, IgA, IgG1, and IgG2b, assaying sera from naive WT and Nfkb1 SSAA/SSAA mice. n = 18 mice per genotype. PEC, peritoneal cavity. *, P

    Article Snippet: GC B cells were stained using GL7-FITC mAb (Ly77, BD), revealed with rabbit anti-FITC (Alexa Fluor 488 Signal-Amplification kit; Invitrogen), IgD-PE (11-26c; eBioscience), and B220-Alexa Fluor 647 (RA3-6B2, BD).

    Techniques: Mouse Assay, Flow Cytometry, Irradiation, Cytometry

    Bcl-xl overexpression does not rescue the TD antibody response of Nfkb1 SSAA/SSAA FM B cells. (A) The fraction of live FM B cells purified from mice of the indicated genotypes was determined as in Fig. 5 C (mean ± SEM). (B) Mixed radiation chimeras were made as in Fig. 2 . LH graph show flow cytometric analysis of splenic B220 + IgM + B cells. Central and RH graphs show anti-NP ELISAs of sera (mean ± SEM) 14 d after NP 27 -CGG immunization. Results are representative of three independent experiments (4–5 mice per genotype). (C) Mean percentages (±SEM) of transferred CD45.2 + cells (left) differentiating to plasmablasts (B220 lo IgM lo IgD − CD138 + CD45.2 + ; middle) and GC B cells (B220 + PNA hi GL7 + CD45.2 + ; right) in the spleens of CD45.1 + WT hosts after 6d immunization with SRBC-HEL or unconjugated SRBC. Results are representative of two independent experiments ( n = 6 mice per condition). Gating strategies used are shown in Fig. S2 (A and B) . **, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKK-induced NF-κB1 p105 proteolysis is critical for B cell antibody responses to T cell–dependent antigen

    doi: 10.1084/jem.20132019

    Figure Lengend Snippet: Bcl-xl overexpression does not rescue the TD antibody response of Nfkb1 SSAA/SSAA FM B cells. (A) The fraction of live FM B cells purified from mice of the indicated genotypes was determined as in Fig. 5 C (mean ± SEM). (B) Mixed radiation chimeras were made as in Fig. 2 . LH graph show flow cytometric analysis of splenic B220 + IgM + B cells. Central and RH graphs show anti-NP ELISAs of sera (mean ± SEM) 14 d after NP 27 -CGG immunization. Results are representative of three independent experiments (4–5 mice per genotype). (C) Mean percentages (±SEM) of transferred CD45.2 + cells (left) differentiating to plasmablasts (B220 lo IgM lo IgD − CD138 + CD45.2 + ; middle) and GC B cells (B220 + PNA hi GL7 + CD45.2 + ; right) in the spleens of CD45.1 + WT hosts after 6d immunization with SRBC-HEL or unconjugated SRBC. Results are representative of two independent experiments ( n = 6 mice per condition). Gating strategies used are shown in Fig. S2 (A and B) . **, P

    Article Snippet: GC B cells were stained using GL7-FITC mAb (Ly77, BD), revealed with rabbit anti-FITC (Alexa Fluor 488 Signal-Amplification kit; Invitrogen), IgD-PE (11-26c; eBioscience), and B220-Alexa Fluor 647 (RA3-6B2, BD).

    Techniques: Over Expression, Purification, Mouse Assay, Flow Cytometry

    Expression pattern of DNMTs in tonsillar B-cell subsets. ( A ) Cartoon illustrating the stages of B-cell development and T-dependent B-cell immune response. B cells are differentiated from hematopoietic stem cells (HSCs) in the bone marrow and released into the periphery upon maturation. Naive B cells encounter antigen-primed T cells in secondary lymphoid organs and enter the germinal center (GC) reaction followed by differentiation into memory B cells or antibody-secreting plasma cells (PCs). ( B ) Purification of B-cell subsets. CD20, IgD, and CD38 cell surface expression were used to distinguish the cell populations: Naive B cells (CD20 + IgD + CD38 lo ), GC B cells (CD20 + IgD − CD38 + ), memory B cells (CD20 + IgD - CD38 − ), and PCs (CD20 lo/+ IgD − CD38 hi ). The percentage of each subset within tonsillar B cells is shown in the FACS plot in the middle panel. Naive, GC, memory, and PC populations were purified from eight individuals for gene expression and DNA methylation profiling on microarrays. The purity of each cell population ( > 90%) was confirmed by flow cytometry analysis of post-sorted cells ( right panel). ( C ) Transcript abundance of DNMT s in B-cell subsets by quantitative RT-PCR. DNMT expression levels are normalized to actin expression level in each cell type. The expression level in one biological replicate of naive B cells is arbitrarily set to one. Error bars denote standard deviation of three biological replicates. ( D ) Expression patterns of DNMT1 and DNMT3A in tonsils. ( Upper left , upper right ) Immunohistochemical staining of DNMT1 at 200× and 400× magnifications, respectively. ( Lower left , lower right ) Immunohistochemical staining of DNMT3A at 200× and 400× magnifications, respectively. ( E ). Scale bar, 50 μm.

    Journal: Genome Research

    Article Title: DNA methylation profiling in human B cells reveals immune regulatory elements and epigenetic plasticity at Alu elements during B-cell activation

    doi: 10.1101/gr.155473.113

    Figure Lengend Snippet: Expression pattern of DNMTs in tonsillar B-cell subsets. ( A ) Cartoon illustrating the stages of B-cell development and T-dependent B-cell immune response. B cells are differentiated from hematopoietic stem cells (HSCs) in the bone marrow and released into the periphery upon maturation. Naive B cells encounter antigen-primed T cells in secondary lymphoid organs and enter the germinal center (GC) reaction followed by differentiation into memory B cells or antibody-secreting plasma cells (PCs). ( B ) Purification of B-cell subsets. CD20, IgD, and CD38 cell surface expression were used to distinguish the cell populations: Naive B cells (CD20 + IgD + CD38 lo ), GC B cells (CD20 + IgD − CD38 + ), memory B cells (CD20 + IgD - CD38 − ), and PCs (CD20 lo/+ IgD − CD38 hi ). The percentage of each subset within tonsillar B cells is shown in the FACS plot in the middle panel. Naive, GC, memory, and PC populations were purified from eight individuals for gene expression and DNA methylation profiling on microarrays. The purity of each cell population ( > 90%) was confirmed by flow cytometry analysis of post-sorted cells ( right panel). ( C ) Transcript abundance of DNMT s in B-cell subsets by quantitative RT-PCR. DNMT expression levels are normalized to actin expression level in each cell type. The expression level in one biological replicate of naive B cells is arbitrarily set to one. Error bars denote standard deviation of three biological replicates. ( D ) Expression patterns of DNMT1 and DNMT3A in tonsils. ( Upper left , upper right ) Immunohistochemical staining of DNMT1 at 200× and 400× magnifications, respectively. ( Lower left , lower right ) Immunohistochemical staining of DNMT3A at 200× and 400× magnifications, respectively. ( E ). Scale bar, 50 μm.

    Article Snippet: The following antibodies were used: PE-IgD (BD Pharmingen), APC-CD38 (eBioscience, HIT2), and FITC-CD20 (eBioscience, 2H7).

    Techniques: Expressing, Purification, FACS, DNA Methylation Assay, Flow Cytometry, Cytometry, Quantitative RT-PCR, Standard Deviation, Immunohistochemistry, Staining

    Phenotypic characterization of circulating B cell subsets in granulomatosis with polyangiitis (GPA) patients and healthy controls (HCs). (A) Flow cytometry gating strategy to distinguish differentiation subsets within peripheral blood CD19 + B cells. IgD + CD27 − transitional/naive B cells, IgD + CD27 + unswitched memory B cells, IgD − CD27 + switched memory B cells and IgD − CD27 − double negative B cells were identified. (B) Relative distribution of distinct CD19 + B cells subsets from HCs (open circles) and GPA patients (gray circles). Horizontal lines indicate median values. Graphs represent data of 17 HCs and 33 GPA patients (** p

    Journal: Frontiers in Immunology

    Article Title: Kv1.3 Channel Blockade Modulates the Effector Function of B Cells in Granulomatosis with Polyangiitis

    doi: 10.3389/fimmu.2017.01205

    Figure Lengend Snippet: Phenotypic characterization of circulating B cell subsets in granulomatosis with polyangiitis (GPA) patients and healthy controls (HCs). (A) Flow cytometry gating strategy to distinguish differentiation subsets within peripheral blood CD19 + B cells. IgD + CD27 − transitional/naive B cells, IgD + CD27 + unswitched memory B cells, IgD − CD27 + switched memory B cells and IgD − CD27 − double negative B cells were identified. (B) Relative distribution of distinct CD19 + B cells subsets from HCs (open circles) and GPA patients (gray circles). Horizontal lines indicate median values. Graphs represent data of 17 HCs and 33 GPA patients (** p

    Article Snippet: Next, 100 µl of the cell suspension was stained with CD19-eFluor450, CD27-APC-eFluor780 (both eBioscience, San Diego, CA, USA), IgD-PE (BD Biosciences, Franklin Lakes, NJ, USA), or the corresponding isotype controls.

    Techniques: Flow Cytometry, Cytometry

    Characterization of T cell receptor and immunoglobulin (Ig) expression on CD32 high CD3 + CD4 + T cells. (A) Expression of TCRαβ on CD32-expressing CD3 + CD4 + T cell populations was measured by flow cytometry and is shown here as median fluorescence intensity. Bar is shown at the mean. (B) Histogram showing TCRαβ staining on CD32-expressing CD3 + CD4 + T cell populations of one sample shown in panel (A) . Fluorescence minus one (FMO) controls are shown as dashed lines and individual populations as solid lines. (C) Percentage expression of Ig was measured on CD3 + CD4 + CD32 high CD20 + cells. Expression of IgD, IgG, and IgM is shown. Expression was measured by flow cytometry, and bar is shown at the mean. (D) Representative staining of data shown in panel (C) .

    Journal: Frontiers in Immunology

    Article Title: CD32-Expressing CD4 T Cells Are Phenotypically Diverse and Can Contain Proviral HIV DNA

    doi: 10.3389/fimmu.2018.00928

    Figure Lengend Snippet: Characterization of T cell receptor and immunoglobulin (Ig) expression on CD32 high CD3 + CD4 + T cells. (A) Expression of TCRαβ on CD32-expressing CD3 + CD4 + T cell populations was measured by flow cytometry and is shown here as median fluorescence intensity. Bar is shown at the mean. (B) Histogram showing TCRαβ staining on CD32-expressing CD3 + CD4 + T cell populations of one sample shown in panel (A) . Fluorescence minus one (FMO) controls are shown as dashed lines and individual populations as solid lines. (C) Percentage expression of Ig was measured on CD3 + CD4 + CD32 high CD20 + cells. Expression of IgD, IgG, and IgM is shown. Expression was measured by flow cytometry, and bar is shown at the mean. (D) Representative staining of data shown in panel (C) .

    Article Snippet: PBMCs were stained with LiveDead Near IR, CD3 AlexaFluor700, CD4 APC, CD32 PE-Cy7, CD14 VioBlue, CD20 FITC and IgD PE (IA6-2), IgG PE (HP6017), or IgM PE (MHM-88) (all BioLegend).

    Techniques: Expressing, Flow Cytometry, Cytometry, Fluorescence, Staining

    B cell depletion by CD20 and CD22 mAbs in NZB/W F 1 and C57BL/6 mice. CD20 ( A ), CD22 ( B ), or IgG2a control mAb (100 μg) was given i.p. to 8–10 wk-old mice with B cell numbers quantified on day 7. B220 + blood, B220 + spleen, B220 + peripheral lymph node, mature recirculating bone marrow (IgD + B220 hi ), marginal zone (B220 + CD21 hi CD1d hi ), and peritoneal B1 (B220 + IgM + CD11b + ) and B2 (B220 + IgM + CD11b − ) cell numbers were determined by immunofluorescence staining with flow cytometric analysis. Representative dot plots are shown for mAb-treated NZB/W F 1 mice with gated B cell percentages indicated. Significant differences between mean (±SEM) values for control mAb-treated (open bars) and CD20 or CD22 mAb-treated (filled bars) mice are indicated; *p

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

    Article Title: Protective and Pathogenic Roles for B Cells During Systemic Autoimmunity in NZB/W F1 Mice

    doi: 10.4049/jimmunol.0902391

    Figure Lengend Snippet: B cell depletion by CD20 and CD22 mAbs in NZB/W F 1 and C57BL/6 mice. CD20 ( A ), CD22 ( B ), or IgG2a control mAb (100 μg) was given i.p. to 8–10 wk-old mice with B cell numbers quantified on day 7. B220 + blood, B220 + spleen, B220 + peripheral lymph node, mature recirculating bone marrow (IgD + B220 hi ), marginal zone (B220 + CD21 hi CD1d hi ), and peritoneal B1 (B220 + IgM + CD11b + ) and B2 (B220 + IgM + CD11b − ) cell numbers were determined by immunofluorescence staining with flow cytometric analysis. Representative dot plots are shown for mAb-treated NZB/W F 1 mice with gated B cell percentages indicated. Significant differences between mean (±SEM) values for control mAb-treated (open bars) and CD20 or CD22 mAb-treated (filled bars) mice are indicated; *p

    Article Snippet: Other reagents included: FITC-, PE-, and PE-cy5-conjugated anti-mouse B220 (CD45R, RA3-6B2), CD5 (53-7.3), CD19 (1D3), CD21/35 (7G6), CD21 (B3B4), CD24 (M1/69), CD43 (S7), and CD93 (AA4.1) mAbs (all from BD Biosciences, San Jose, CA); CD11b (M170) and CD1d (1B1; from eBioscience, San Diego, CA) mAbs; and polyclonal goat anti-IgM and –IgD Abs (Southern Biotechnology Associates).

    Techniques: Mouse Assay, Immunofluorescence, Staining, Flow Cytometry

    Fab-PLA of the nanoscale proximities of the IgM-BCR and IgD-BCR to the lipid raft marker CD52. ( A ) Representative microscopic images of the IgM:CD52 Fab-PLA on resting (upper) or NIP-BSA stimulated (lower) TKO-M cells (left) or the IgD:CD52 Fab-PLA on resting (upper) or NIP-BSA stimulated (lower) TKO-D cells (right). Scale bar: 5 μm. ( B ) Quantified results of IgM:CD52 (left) and IgD:CD52 (right) Fab-PLA of resting and stimulated TKO-M or TKO-D cells. Data represent the mean and SEM of a minimum of three independent experiments. For each experiment, PLA signals (counts per cell) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting (IgD:CD52) or NIP-BSA stimulated (IgM:CD52) cells. DOI: http://dx.doi.org/10.7554/eLife.02069.013

    Journal: eLife

    Article Title: B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk

    doi: 10.7554/eLife.02069

    Figure Lengend Snippet: Fab-PLA of the nanoscale proximities of the IgM-BCR and IgD-BCR to the lipid raft marker CD52. ( A ) Representative microscopic images of the IgM:CD52 Fab-PLA on resting (upper) or NIP-BSA stimulated (lower) TKO-M cells (left) or the IgD:CD52 Fab-PLA on resting (upper) or NIP-BSA stimulated (lower) TKO-D cells (right). Scale bar: 5 μm. ( B ) Quantified results of IgM:CD52 (left) and IgD:CD52 (right) Fab-PLA of resting and stimulated TKO-M or TKO-D cells. Data represent the mean and SEM of a minimum of three independent experiments. For each experiment, PLA signals (counts per cell) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting (IgD:CD52) or NIP-BSA stimulated (IgM:CD52) cells. DOI: http://dx.doi.org/10.7554/eLife.02069.013

    Article Snippet: For PLA probes against specific targets, the following unlabelled antibodies were used: IgD (11-26c.2a, SouthernBiotech, Birmingham, AL), IgD (AMS9.1; Santa Cruz Biotechnology, Dallas, TX), IgM (R33.24.12, in house hybridoma culture), IgM (rabbit anti-mouse µHC; Rockland Immunochemicals, Gilbertsville, PA), IgM (1B4B1; SouthernBiotech), Lambda light chain (JC5-1; SouthernBiotech), Kappa light chain (187.1; SouthernBiotech), CD19 (6D5; AbD Serotec, Düsseldorf, Germany) and CD20 (AISB12; eBioscience).

    Techniques: Proximity Ligation Assay, Marker

    Nanoscale IgM-BCR and IgD-BCR dissociation and coreceptor reorganization on B cells expressing only one BCR isotype. ( A – C ) Representative microscopic images (left) and quantified results (right) of the IgM:IgM ( A ), the IgM:CD19 ( B ), and the IgM:CD20 ( C ) Fab-PLA of resting or activated TKO-M cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate and the F-actin inhibitor Lat-A. ( D – F ) Representative microscopic images (left) and quantified results (right) of the IgD:IgD ( D ), the IgD:CD19 ( E ), and the IgD:CD20 ( F ) Fab-PLA of resting or activated TKO-D cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate and the F-actin inhibitor Lat-A. Scale bar: 5 μm. The quantified data represent the mean and SEM of a minimum of three independent experiments. For each experiment, PLA signals (counts per cell) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting ( A , D , E , F ) or NIP-BSA stimulated ( B and C ) cells. DOI: http://dx.doi.org/10.7554/eLife.02069.012

    Journal: eLife

    Article Title: B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk

    doi: 10.7554/eLife.02069

    Figure Lengend Snippet: Nanoscale IgM-BCR and IgD-BCR dissociation and coreceptor reorganization on B cells expressing only one BCR isotype. ( A – C ) Representative microscopic images (left) and quantified results (right) of the IgM:IgM ( A ), the IgM:CD19 ( B ), and the IgM:CD20 ( C ) Fab-PLA of resting or activated TKO-M cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate and the F-actin inhibitor Lat-A. ( D – F ) Representative microscopic images (left) and quantified results (right) of the IgD:IgD ( D ), the IgD:CD19 ( E ), and the IgD:CD20 ( F ) Fab-PLA of resting or activated TKO-D cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate and the F-actin inhibitor Lat-A. Scale bar: 5 μm. The quantified data represent the mean and SEM of a minimum of three independent experiments. For each experiment, PLA signals (counts per cell) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting ( A , D , E , F ) or NIP-BSA stimulated ( B and C ) cells. DOI: http://dx.doi.org/10.7554/eLife.02069.012

    Article Snippet: For PLA probes against specific targets, the following unlabelled antibodies were used: IgD (11-26c.2a, SouthernBiotech, Birmingham, AL), IgD (AMS9.1; Santa Cruz Biotechnology, Dallas, TX), IgM (R33.24.12, in house hybridoma culture), IgM (rabbit anti-mouse µHC; Rockland Immunochemicals, Gilbertsville, PA), IgM (1B4B1; SouthernBiotech), Lambda light chain (JC5-1; SouthernBiotech), Kappa light chain (187.1; SouthernBiotech), CD19 (6D5; AbD Serotec, Düsseldorf, Germany) and CD20 (AISB12; eBioscience).

    Techniques: Expressing, Proximity Ligation Assay

    Fab-PLA detects IgD-BCR oligomers on the S2 cell surface. ( A ) Schematic drawing of wild type and double-mutant IgD-BCRs (δm transmembrane mutations and removal of the S–S bridge of the Igα/Igβ heterodimer) and their expression on transfected S2 cells analyzed by FACScan with a fluorescent 1NIP-peptide. Positively transfected S2 cells are indicated by the expression of a cotransfected GFP vector. The double-positive (GFP+, BCR+) S2 cell population, indicated by red square, were sorted and used for Fab-PLA. ( B ) Confocal microscopy analysis of the IgD:IgD Fab-PLA reaction of GFP+ S2 cells expressing wild type (left panel) or double-mutant (right panel) IgD-BCR. PLA signals are shown as red dots and nuclei were visualized by DAPI staining (blue). Scale bar: 5 μm. ( C ) Quantification of IgD:IgD Fab-PLA (each dot represents the amounts of Fab-PLA signals per S2 cell). The data were analyzed by the mann-whitney test and the median values are shown as red line. DOI: http://dx.doi.org/10.7554/eLife.02069.006

    Journal: eLife

    Article Title: B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk

    doi: 10.7554/eLife.02069

    Figure Lengend Snippet: Fab-PLA detects IgD-BCR oligomers on the S2 cell surface. ( A ) Schematic drawing of wild type and double-mutant IgD-BCRs (δm transmembrane mutations and removal of the S–S bridge of the Igα/Igβ heterodimer) and their expression on transfected S2 cells analyzed by FACScan with a fluorescent 1NIP-peptide. Positively transfected S2 cells are indicated by the expression of a cotransfected GFP vector. The double-positive (GFP+, BCR+) S2 cell population, indicated by red square, were sorted and used for Fab-PLA. ( B ) Confocal microscopy analysis of the IgD:IgD Fab-PLA reaction of GFP+ S2 cells expressing wild type (left panel) or double-mutant (right panel) IgD-BCR. PLA signals are shown as red dots and nuclei were visualized by DAPI staining (blue). Scale bar: 5 μm. ( C ) Quantification of IgD:IgD Fab-PLA (each dot represents the amounts of Fab-PLA signals per S2 cell). The data were analyzed by the mann-whitney test and the median values are shown as red line. DOI: http://dx.doi.org/10.7554/eLife.02069.006

    Article Snippet: For PLA probes against specific targets, the following unlabelled antibodies were used: IgD (11-26c.2a, SouthernBiotech, Birmingham, AL), IgD (AMS9.1; Santa Cruz Biotechnology, Dallas, TX), IgM (R33.24.12, in house hybridoma culture), IgM (rabbit anti-mouse µHC; Rockland Immunochemicals, Gilbertsville, PA), IgM (1B4B1; SouthernBiotech), Lambda light chain (JC5-1; SouthernBiotech), Kappa light chain (187.1; SouthernBiotech), CD19 (6D5; AbD Serotec, Düsseldorf, Germany) and CD20 (AISB12; eBioscience).

    Techniques: Proximity Ligation Assay, Mutagenesis, Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Staining, MANN-WHITNEY

    Similar binding efficiency of Fab-PLA probes on resting and stimulated B cells. ( A ) Schematic drawing of Fab-PLA probes and control probes used for the binding assay. ( B ) FACScan analysis of anti-IgM Fab-PLA plus (upper left) and minus (lower left), anti-IgD Fab-PLA plus (upper middle) and minus (lower middle), anti-CD19 Fab-PLA plus (upper right) and anti-CD20 Fab-PLA minus (lower right) probes binding on resting or differently (NIP-BSA, Pervanadate, Latrunculin-A), stimulated B1-8 B cells. ( C – F ) Nanoscale proximity of heavy chain (HC) and light chain (LC) examined by Fab-PLA remained unaltered in TKO ( C and D ) and B1-8 B cells ( E and F ) upon activation with different stimuli. Representative microscopic images (left) and quantified results (right) show the proximity of ( C and E ) μm HC or ( D and F ) δm HC to λ LC of resting or activated B cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate (Perv) and the F-actin inhibitor Lat-A. Scale bar: 5 μm. Data represent the mean and SEM of four independent experiments. For each experiment, PLA signals (counts per cell) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting B cells. DOI: http://dx.doi.org/10.7554/eLife.02069.005

    Journal: eLife

    Article Title: B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk

    doi: 10.7554/eLife.02069

    Figure Lengend Snippet: Similar binding efficiency of Fab-PLA probes on resting and stimulated B cells. ( A ) Schematic drawing of Fab-PLA probes and control probes used for the binding assay. ( B ) FACScan analysis of anti-IgM Fab-PLA plus (upper left) and minus (lower left), anti-IgD Fab-PLA plus (upper middle) and minus (lower middle), anti-CD19 Fab-PLA plus (upper right) and anti-CD20 Fab-PLA minus (lower right) probes binding on resting or differently (NIP-BSA, Pervanadate, Latrunculin-A), stimulated B1-8 B cells. ( C – F ) Nanoscale proximity of heavy chain (HC) and light chain (LC) examined by Fab-PLA remained unaltered in TKO ( C and D ) and B1-8 B cells ( E and F ) upon activation with different stimuli. Representative microscopic images (left) and quantified results (right) show the proximity of ( C and E ) μm HC or ( D and F ) δm HC to λ LC of resting or activated B cells stimulated for 5 min with the antigen NIP-BSA, the oxidant pervanadate (Perv) and the F-actin inhibitor Lat-A. Scale bar: 5 μm. Data represent the mean and SEM of four independent experiments. For each experiment, PLA signals (counts per cell) of each sample were counted from a minimum of 1000 cells and were then normalized to the PLA signal of the resting B cells. DOI: http://dx.doi.org/10.7554/eLife.02069.005

    Article Snippet: For PLA probes against specific targets, the following unlabelled antibodies were used: IgD (11-26c.2a, SouthernBiotech, Birmingham, AL), IgD (AMS9.1; Santa Cruz Biotechnology, Dallas, TX), IgM (R33.24.12, in house hybridoma culture), IgM (rabbit anti-mouse µHC; Rockland Immunochemicals, Gilbertsville, PA), IgM (1B4B1; SouthernBiotech), Lambda light chain (JC5-1; SouthernBiotech), Kappa light chain (187.1; SouthernBiotech), CD19 (6D5; AbD Serotec, Düsseldorf, Germany) and CD20 (AISB12; eBioscience).

    Techniques: Binding Assay, Proximity Ligation Assay, Activation Assay

    Fab-PLA study of the nanoscale organization of the IgM-BCR and the IgD-BCR on resting and activated B cells. ( A ) The IgM:IgM (upper) and IgD:IgD (lower) proximity of antigen receptors on resting or activated B1-8 splenic B cells were examined by Fab-PLA and shown as representative microscopic images (left) and quantified results (right). ( B – D ) Quantified Fab-PLA results indicate the IgM:IgM (upper) and IgD:IgD (lower) proximity on resting or activated TKO-MD cells ( B ), B1-8 splenic B cells ( C ) and human IgM+IgD+ naïve B cells isolated from peripheral blood ( D ). Scale bar: 5 μm. Quantified data represent the mean and SEM of a minimum of four independent experiments. For each experiment, PLA signals (counts per cell) of each sample were counted from a minimum of 100 cells and were then normalized to the PLA signals of the resting cells. DOI: http://dx.doi.org/10.7554/eLife.02069.007

    Journal: eLife

    Article Title: B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk

    doi: 10.7554/eLife.02069

    Figure Lengend Snippet: Fab-PLA study of the nanoscale organization of the IgM-BCR and the IgD-BCR on resting and activated B cells. ( A ) The IgM:IgM (upper) and IgD:IgD (lower) proximity of antigen receptors on resting or activated B1-8 splenic B cells were examined by Fab-PLA and shown as representative microscopic images (left) and quantified results (right). ( B – D ) Quantified Fab-PLA results indicate the IgM:IgM (upper) and IgD:IgD (lower) proximity on resting or activated TKO-MD cells ( B ), B1-8 splenic B cells ( C ) and human IgM+IgD+ naïve B cells isolated from peripheral blood ( D ). Scale bar: 5 μm. Quantified data represent the mean and SEM of a minimum of four independent experiments. For each experiment, PLA signals (counts per cell) of each sample were counted from a minimum of 100 cells and were then normalized to the PLA signals of the resting cells. DOI: http://dx.doi.org/10.7554/eLife.02069.007

    Article Snippet: For PLA probes against specific targets, the following unlabelled antibodies were used: IgD (11-26c.2a, SouthernBiotech, Birmingham, AL), IgD (AMS9.1; Santa Cruz Biotechnology, Dallas, TX), IgM (R33.24.12, in house hybridoma culture), IgM (rabbit anti-mouse µHC; Rockland Immunochemicals, Gilbertsville, PA), IgM (1B4B1; SouthernBiotech), Lambda light chain (JC5-1; SouthernBiotech), Kappa light chain (187.1; SouthernBiotech), CD19 (6D5; AbD Serotec, Düsseldorf, Germany) and CD20 (AISB12; eBioscience).

    Techniques: Proximity Ligation Assay, Isolation

    Phenotypic characterization of B cells from SW HEL mice. (A) Spleen cells from 10–14-wk-old NB6, LC2, V H 10 tar +/− , SW HEL (V H 10 tar +/− × LC2), and MD4 Ig Tg mice were harvested and stained with anti–B220-APC, HEL plus anti–HEL-FITC, anti–IgD-PE, and anti–IgM-biotin plus SA-PerCP. Cells were analyzed by flow cytometry and the frequency of HEL + (top box) and HEL − (bottom box) B cells in each density plot was indicated. Values from each mouse are representative of more than five independent experiments. (B) Spleen cells were stained as for A. IgM and IgD expression data are displayed for all B220 + B cells (NB6 and MD4, top) or specifically for HEL + and HEL − B cells (SW HEL , bottom) using the gates shown in A. Numbers indicate the proportions of gated cells within the indicated quadrants. Data are representative of more than five independent experiments. (C) Cells were isolated by peritoneal lavage from the indicated mice and stained with anti–B220-APC, HEL plus anti–HEL-FITC, anti–IgM-PE, and anti–CD5-biotin plus SA-PerCP. Cells were analyzed by flow cytometry and gated on live cells that were B220 + and/or IgM + to include both the B1 and B2 cell populations. For SW HEL mice the peritoneal cavity B cells were further gated into HEL + and HEL − subpopulations. Numbers indicate the proportion of gated cells in the B220 hi CD5 − window. Data are representative of three independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: B Cell Receptor-independent Stimuli Trigger Immunoglobulin (Ig) Class Switch Recombination and Production of IgG Autoantibodies by Anergic Self-Reactive B Cells

    doi: 10.1084/jem.20022144

    Figure Lengend Snippet: Phenotypic characterization of B cells from SW HEL mice. (A) Spleen cells from 10–14-wk-old NB6, LC2, V H 10 tar +/− , SW HEL (V H 10 tar +/− × LC2), and MD4 Ig Tg mice were harvested and stained with anti–B220-APC, HEL plus anti–HEL-FITC, anti–IgD-PE, and anti–IgM-biotin plus SA-PerCP. Cells were analyzed by flow cytometry and the frequency of HEL + (top box) and HEL − (bottom box) B cells in each density plot was indicated. Values from each mouse are representative of more than five independent experiments. (B) Spleen cells were stained as for A. IgM and IgD expression data are displayed for all B220 + B cells (NB6 and MD4, top) or specifically for HEL + and HEL − B cells (SW HEL , bottom) using the gates shown in A. Numbers indicate the proportions of gated cells within the indicated quadrants. Data are representative of more than five independent experiments. (C) Cells were isolated by peritoneal lavage from the indicated mice and stained with anti–B220-APC, HEL plus anti–HEL-FITC, anti–IgM-PE, and anti–CD5-biotin plus SA-PerCP. Cells were analyzed by flow cytometry and gated on live cells that were B220 + and/or IgM + to include both the B1 and B2 cell populations. For SW HEL mice the peritoneal cavity B cells were further gated into HEL + and HEL − subpopulations. Numbers indicate the proportion of gated cells in the B220 hi CD5 − window. Data are representative of three independent experiments.

    Article Snippet: Anti–IgM-PE (1B4B1) and IgD-PE ( - ) were purchased from Southern Biotechnology Associates, Inc. HyHEL5, HyHEL9, anti-IgM (Bet-2), IgMa (RS-3.1), and IgDa (AMS-15.1) mAbs were purified from hybridoma supernatants and either biotinylated or conjugated to FITC as previously described ( ).

    Techniques: Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing, Isolation