cd21  (Thermo Fisher)


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    Name:
    CD21 CR2 Monoclonal Antibody 7E9
    Description:
    CD21 CR2 Monoclonal Antibody for Flow
    Catalog Number:
    a14921
    Price:
    None
    Applications:
    Cell Analysis|Flow Antibodies for Cytokine, Chemokine, Growth Factor & Hormone Receptors|Flow Cytometry Antibodies & Secondary Detection|Flow Cytometry Antibodies for CD Markers|Flow Cytometry
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher cd21
    IL35 is upregulated in CD4 + T cells and B cells in PDA. (A) Representative flow cytometry plots of CD4 + and CD19 + cells isolated from the spleens of WT or KC mice at 4 months of age. Cells were processed for intracellular staining with anti-p35 and anti-Ebi3. Percent of p35 + Ebi3 + CD4 + T cells and p35 + Ebi3 + CD19 + B cells is indicated. (B) Percentage of IL35 + splenic T cells, B cells, or myeloid cells in WT (n=12) or splenic and intratumoral IL35 + T cells, B cells, or myeloid cells KC mice (n=12). (C) Percentage of IL35 + splenic T cells, B cells, or myeloid cells in WT or splenic and intratumoral IL35 + splenic T cells, B cells, or myeloid cells in mice orthotopically injected with KPC 4662 cells and collected 3 weeks post-tumor cell injection. (D) Representative flow cytometry plots of intratumoral B conventional (Bcon; CD19 + <t>CD21</t> + CD5 – CD1d – ) and B regulatory cells (Breg; CD19 + CD21 hi CD5 + CD1d hi ) from KC mice at 4 months of age or mice orthotopically injected with KPC cells and collected 3 weeks post-tumor cell injection. Cells were analyzed by staining with anti-p35 antibody. Percent of p35 + Bcon or Breg cells is indicated. (E) Representative flow cytometry plots of intratumoral CD4 + Foxp3 – T cells and CD4 + Foxp3 + Treg cells from KC mice at 4 months of age. Cells were analyzed by staining with anti-p35 antibody. Percent of p35 + CD4 + Foxp3 – or Treg cells is indicated. (F) Representative flow cytometry plots of intratumoral CD4 + Foxp3 – T cells and Tregs from mice orthotopically injected with KPC cells and collected 3 weeks post-tumor cell injection. Cells were analyzed by staining with anti-p35 antibody. Percent of p35 + CD4 + Foxp3 – or Treg cells is indicated. Error bars indicate SEM. p -values were calculated using Student’s t-test (unpaired, two-tailed); NS – not significant, * p
    CD21 CR2 Monoclonal Antibody for Flow
    https://www.bioz.com/result/cd21/product/Thermo Fisher
    Average 96 stars, based on 66 article reviews
    Price from $9.99 to $1999.99
    cd21 - by Bioz Stars, 2020-11
    96/100 stars

    Images

    1) Product Images from "IL35 hinders endogenous antitumor T-cell immunity and responsiveness to immunotherapy in pancreatic cancer"

    Article Title: IL35 hinders endogenous antitumor T-cell immunity and responsiveness to immunotherapy in pancreatic cancer

    Journal: Cancer immunology research

    doi: 10.1158/2326-6066.CIR-17-0710

    IL35 is upregulated in CD4 + T cells and B cells in PDA. (A) Representative flow cytometry plots of CD4 + and CD19 + cells isolated from the spleens of WT or KC mice at 4 months of age. Cells were processed for intracellular staining with anti-p35 and anti-Ebi3. Percent of p35 + Ebi3 + CD4 + T cells and p35 + Ebi3 + CD19 + B cells is indicated. (B) Percentage of IL35 + splenic T cells, B cells, or myeloid cells in WT (n=12) or splenic and intratumoral IL35 + T cells, B cells, or myeloid cells KC mice (n=12). (C) Percentage of IL35 + splenic T cells, B cells, or myeloid cells in WT or splenic and intratumoral IL35 + splenic T cells, B cells, or myeloid cells in mice orthotopically injected with KPC 4662 cells and collected 3 weeks post-tumor cell injection. (D) Representative flow cytometry plots of intratumoral B conventional (Bcon; CD19 + CD21 + CD5 – CD1d – ) and B regulatory cells (Breg; CD19 + CD21 hi CD5 + CD1d hi ) from KC mice at 4 months of age or mice orthotopically injected with KPC cells and collected 3 weeks post-tumor cell injection. Cells were analyzed by staining with anti-p35 antibody. Percent of p35 + Bcon or Breg cells is indicated. (E) Representative flow cytometry plots of intratumoral CD4 + Foxp3 – T cells and CD4 + Foxp3 + Treg cells from KC mice at 4 months of age. Cells were analyzed by staining with anti-p35 antibody. Percent of p35 + CD4 + Foxp3 – or Treg cells is indicated. (F) Representative flow cytometry plots of intratumoral CD4 + Foxp3 – T cells and Tregs from mice orthotopically injected with KPC cells and collected 3 weeks post-tumor cell injection. Cells were analyzed by staining with anti-p35 antibody. Percent of p35 + CD4 + Foxp3 – or Treg cells is indicated. Error bars indicate SEM. p -values were calculated using Student’s t-test (unpaired, two-tailed); NS – not significant, * p
    Figure Legend Snippet: IL35 is upregulated in CD4 + T cells and B cells in PDA. (A) Representative flow cytometry plots of CD4 + and CD19 + cells isolated from the spleens of WT or KC mice at 4 months of age. Cells were processed for intracellular staining with anti-p35 and anti-Ebi3. Percent of p35 + Ebi3 + CD4 + T cells and p35 + Ebi3 + CD19 + B cells is indicated. (B) Percentage of IL35 + splenic T cells, B cells, or myeloid cells in WT (n=12) or splenic and intratumoral IL35 + T cells, B cells, or myeloid cells KC mice (n=12). (C) Percentage of IL35 + splenic T cells, B cells, or myeloid cells in WT or splenic and intratumoral IL35 + splenic T cells, B cells, or myeloid cells in mice orthotopically injected with KPC 4662 cells and collected 3 weeks post-tumor cell injection. (D) Representative flow cytometry plots of intratumoral B conventional (Bcon; CD19 + CD21 + CD5 – CD1d – ) and B regulatory cells (Breg; CD19 + CD21 hi CD5 + CD1d hi ) from KC mice at 4 months of age or mice orthotopically injected with KPC cells and collected 3 weeks post-tumor cell injection. Cells were analyzed by staining with anti-p35 antibody. Percent of p35 + Bcon or Breg cells is indicated. (E) Representative flow cytometry plots of intratumoral CD4 + Foxp3 – T cells and CD4 + Foxp3 + Treg cells from KC mice at 4 months of age. Cells were analyzed by staining with anti-p35 antibody. Percent of p35 + CD4 + Foxp3 – or Treg cells is indicated. (F) Representative flow cytometry plots of intratumoral CD4 + Foxp3 – T cells and Tregs from mice orthotopically injected with KPC cells and collected 3 weeks post-tumor cell injection. Cells were analyzed by staining with anti-p35 antibody. Percent of p35 + CD4 + Foxp3 – or Treg cells is indicated. Error bars indicate SEM. p -values were calculated using Student’s t-test (unpaired, two-tailed); NS – not significant, * p

    Techniques Used: Flow Cytometry, Cytometry, Isolation, Mouse Assay, Staining, Injection, Two Tailed Test

    2) Product Images from "α-Galactosylceramide stimulates splenic lymphocyte proliferation in vitro and increases antibody production in vivo in late neonatal-age mice"

    Article Title: α-Galactosylceramide stimulates splenic lymphocyte proliferation in vitro and increases antibody production in vivo in late neonatal-age mice

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.12447

    Alpha-galactosylceramide (αGalCer) increases proliferation of follicular B cells. Carboxyfluorescein succinimidyl ester (CFSE)-labelled splenocytes were cultured as in Fig. and stained with anti-CD19, CD21 and CD23. (a) αGalCer
    Figure Legend Snippet: Alpha-galactosylceramide (αGalCer) increases proliferation of follicular B cells. Carboxyfluorescein succinimidyl ester (CFSE)-labelled splenocytes were cultured as in Fig. and stained with anti-CD19, CD21 and CD23. (a) αGalCer

    Techniques Used: Cell Culture, Staining

    3) Product Images from "Autologous Splenocyte Reinfusion Improves Antibody-Mediated Immune Response to the 23-Valent Pneumococcal Polysaccharide-Based Vaccine in Splenectomized Mice"

    Article Title: Autologous Splenocyte Reinfusion Improves Antibody-Mediated Immune Response to the 23-Valent Pneumococcal Polysaccharide-Based Vaccine in Splenectomized Mice

    Journal: Biomolecules

    doi: 10.3390/biom10050704

    Summary of the procedure for restoring the innate humoral antibody-mediated immunity responsive to Pneumococcal polyvalent 23 serotypes (Pneumovax ® 23) through autologous splenocyte reinfusion upon splenectomy mice. ( A ) Flow chart of the procedure—both IgM immune response after initial vaccination (Group A) and IgG immune response following revaccination (Group B). Each group had three separate subgroups (1, 2, and 3). Subgroup 1 (positive control subgroup) were the sham control mice, subgroup 2 (negative control subgroup) consisted of splenectomized mice (−SL), and subgroup 3 (experimental subgroup) consisted of splenectomized mice with autologous splenic lymphocytes (+SL) reinfusion. Inset: micro hematocrit capillary tubes were used to separate serum from blood cells, derived from blood draw via retro-orbital venous plexus. ( B ) Representatives of FACS analysis of splenic lymphocytes. Fractions of the cells (#062513) were used for FACS analysis with fluorophore-conjugated antibodies against CD3, CD19, CD21, CD35, CD80, respectively, with corresponding isotypes as controls as described in the manufacturer’s Manuel (eBioscience, Affymetrix, Inc., Santa Clara, CA, USA), as described in Methods. Specifically, CD80-FITC (for mature B cells), CD19-PE (pan-marker for global B cells), CD3-eFluor660 (for T cells), CD21-APC (for naïve B cells) were used to determine the purity of cell types of the splenic lymphocyte preparation. As the majority of splenic lymphocytes are B cells and the other B cells are located in lymph nodes and circulation, both CD80 for mature B cells and CD21 for naïve B cells were accessed (Note: Gate R1, 79.4% of the cells were used for analysis regions that illustrated as the analysis histograms).
    Figure Legend Snippet: Summary of the procedure for restoring the innate humoral antibody-mediated immunity responsive to Pneumococcal polyvalent 23 serotypes (Pneumovax ® 23) through autologous splenocyte reinfusion upon splenectomy mice. ( A ) Flow chart of the procedure—both IgM immune response after initial vaccination (Group A) and IgG immune response following revaccination (Group B). Each group had three separate subgroups (1, 2, and 3). Subgroup 1 (positive control subgroup) were the sham control mice, subgroup 2 (negative control subgroup) consisted of splenectomized mice (−SL), and subgroup 3 (experimental subgroup) consisted of splenectomized mice with autologous splenic lymphocytes (+SL) reinfusion. Inset: micro hematocrit capillary tubes were used to separate serum from blood cells, derived from blood draw via retro-orbital venous plexus. ( B ) Representatives of FACS analysis of splenic lymphocytes. Fractions of the cells (#062513) were used for FACS analysis with fluorophore-conjugated antibodies against CD3, CD19, CD21, CD35, CD80, respectively, with corresponding isotypes as controls as described in the manufacturer’s Manuel (eBioscience, Affymetrix, Inc., Santa Clara, CA, USA), as described in Methods. Specifically, CD80-FITC (for mature B cells), CD19-PE (pan-marker for global B cells), CD3-eFluor660 (for T cells), CD21-APC (for naïve B cells) were used to determine the purity of cell types of the splenic lymphocyte preparation. As the majority of splenic lymphocytes are B cells and the other B cells are located in lymph nodes and circulation, both CD80 for mature B cells and CD21 for naïve B cells were accessed (Note: Gate R1, 79.4% of the cells were used for analysis regions that illustrated as the analysis histograms).

    Techniques Used: Mouse Assay, Positive Control, Negative Control, Derivative Assay, FACS, Marker

    4) Product Images from "In Vivo Suppression of Autophagy via Lentiviral shRNA Targeting Atg5 Improves Lupus-Like Syndrome"

    Article Title: In Vivo Suppression of Autophagy via Lentiviral shRNA Targeting Atg5 Improves Lupus-Like Syndrome

    Journal: BioMed Research International

    doi: 10.1155/2020/8959726

    Silencing Atg5 reduced the number of several immune cells in the lymph nodes of Trem-1 −/− .lpr mice. shLuc- or shAtg5-lentivirus-infected mice were euthanized at 38-40 weeks old. Single-cell suspension from the lymph nodes was obtained by passing them through a nylon filter. (a) Single-cell suspension was stained with AO and analyzed using flow cytometry to determine the autophagy level (AVOs (%)). (b) Total lymph node cell numbers were counted and compared. (c, d, e) The percentages (left) and cell numbers (right) of different immune cell subsets were evaluated by flow cytometry using various cell-specific markers, including total B cell (CD19 + ), total T cell (CD3 + ), mDC (CD11c + B220 − ), pDC (CD11c + B220 + ), T1+T2 B (transitional B cell, CD19 + CD21 -/low CD23 -/low ), FoB (follicular B cell, CD19 + CD21 + CD23 hi ), MZB (marginal zone B cell, CD19 + CD21 hi CD23 + ), PC (plasma cell, CD138 + ), DN T (double-negative T cell, CD3 + CD4 − CD8 − , DP T (double-positive T cell, CD3 + CD4 + CD8 + ), T helper cell (CD3 + CD4 + ), cytotoxic T cell (CD3 + CD8 + ), and regulatory T cell (CD3 + CD4 + CD25 + FoxP3 + ). Mean values were shown by a bar.
    Figure Legend Snippet: Silencing Atg5 reduced the number of several immune cells in the lymph nodes of Trem-1 −/− .lpr mice. shLuc- or shAtg5-lentivirus-infected mice were euthanized at 38-40 weeks old. Single-cell suspension from the lymph nodes was obtained by passing them through a nylon filter. (a) Single-cell suspension was stained with AO and analyzed using flow cytometry to determine the autophagy level (AVOs (%)). (b) Total lymph node cell numbers were counted and compared. (c, d, e) The percentages (left) and cell numbers (right) of different immune cell subsets were evaluated by flow cytometry using various cell-specific markers, including total B cell (CD19 + ), total T cell (CD3 + ), mDC (CD11c + B220 − ), pDC (CD11c + B220 + ), T1+T2 B (transitional B cell, CD19 + CD21 -/low CD23 -/low ), FoB (follicular B cell, CD19 + CD21 + CD23 hi ), MZB (marginal zone B cell, CD19 + CD21 hi CD23 + ), PC (plasma cell, CD138 + ), DN T (double-negative T cell, CD3 + CD4 − CD8 − , DP T (double-positive T cell, CD3 + CD4 + CD8 + ), T helper cell (CD3 + CD4 + ), cytotoxic T cell (CD3 + CD8 + ), and regulatory T cell (CD3 + CD4 + CD25 + FoxP3 + ). Mean values were shown by a bar.

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

    5) Product Images from "B cell expression of the SH2-containing inositol 5-phosphatase (SHIP-1) is required to establish anergy to high affinity, proteinacious autoantigens"

    Article Title: B cell expression of the SH2-containing inositol 5-phosphatase (SHIP-1) is required to establish anergy to high affinity, proteinacious autoantigens

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2015.06.007

    B cells from SHIP-deficient MD4.ML-5 mice are ignorant of HEL A. IgM and IgD expression by ex-vivo splenic B cells from SHIP-sufficient MD4.ML-5 (black), SHIP-deficient MD4.ML-5 (gray), MD4 (dashed black), SHIP-1 deficient (dashed gray) and T cells (gray shaded) is shown. B. Splenic B cells from SHIP-sufficient MD4.ML-5 (black), SHIP-deficient MD4.ML-5 (gray) and MD4 SHIP-sufficient control mice (dashed black) were Indo1-AM loaded and stained with non-stimulating antibodies specific to B220, CD21 and CD23. Follicular B cells were identified by gating on B220 + , CD23 hi , CD21 mid cells and intracellular free calcium of these cells was monitored before and after stimulation with 15 ng/mL HEL. Receptor occupancy of each population was assessed immediately ex vivo and is noted adjacent to trace. C. Anti-HEL antibody levels were determined by ELISA of serum from SHIP-sufficient MD4.ML-5 (square), SHIP-deficient MD4.ML-5 (circle) and C57BL/6 (triangle) mice. D. Anti-HEL antibody secreting cells (ASC) were measured by ELISPOT analysis of splenocytes from MD4.ML-5 mice in which B cells were SHIP-sufficient (squares) or SHIP-deficient (circles). Shaded area reflects the lower limit of detection of the assay. E. IgM a anti-HEL:HEL complexes were measured by ELISA of serum from SHIP-sufficient MD4.ML-5 (squares), SHIP-deficient MD4.ML-5 (circles) and C57Bl/6 mice (triangles). F. BCR occupancy by HEL in splenic B cells from SHIP-sufficient MD4.ML-5 (left panel) or SHIP-deficient MD4.ML-5 (right panel) mice. Shown are histograms illustrating occupancy of experimental populations (gray), compared to internal 0% occupancy (gray shaded) and 100% occupancy (black) controls. Bar graph below shows quantification of RO distribution for all mice in experiment; SHIP-sufficient (black), SHIP-deficient (gray). Characterization was repeated twice with at least three mice per experimental group.
    Figure Legend Snippet: B cells from SHIP-deficient MD4.ML-5 mice are ignorant of HEL A. IgM and IgD expression by ex-vivo splenic B cells from SHIP-sufficient MD4.ML-5 (black), SHIP-deficient MD4.ML-5 (gray), MD4 (dashed black), SHIP-1 deficient (dashed gray) and T cells (gray shaded) is shown. B. Splenic B cells from SHIP-sufficient MD4.ML-5 (black), SHIP-deficient MD4.ML-5 (gray) and MD4 SHIP-sufficient control mice (dashed black) were Indo1-AM loaded and stained with non-stimulating antibodies specific to B220, CD21 and CD23. Follicular B cells were identified by gating on B220 + , CD23 hi , CD21 mid cells and intracellular free calcium of these cells was monitored before and after stimulation with 15 ng/mL HEL. Receptor occupancy of each population was assessed immediately ex vivo and is noted adjacent to trace. C. Anti-HEL antibody levels were determined by ELISA of serum from SHIP-sufficient MD4.ML-5 (square), SHIP-deficient MD4.ML-5 (circle) and C57BL/6 (triangle) mice. D. Anti-HEL antibody secreting cells (ASC) were measured by ELISPOT analysis of splenocytes from MD4.ML-5 mice in which B cells were SHIP-sufficient (squares) or SHIP-deficient (circles). Shaded area reflects the lower limit of detection of the assay. E. IgM a anti-HEL:HEL complexes were measured by ELISA of serum from SHIP-sufficient MD4.ML-5 (squares), SHIP-deficient MD4.ML-5 (circles) and C57Bl/6 mice (triangles). F. BCR occupancy by HEL in splenic B cells from SHIP-sufficient MD4.ML-5 (left panel) or SHIP-deficient MD4.ML-5 (right panel) mice. Shown are histograms illustrating occupancy of experimental populations (gray), compared to internal 0% occupancy (gray shaded) and 100% occupancy (black) controls. Bar graph below shows quantification of RO distribution for all mice in experiment; SHIP-sufficient (black), SHIP-deficient (gray). Characterization was repeated twice with at least three mice per experimental group.

    Techniques Used: Mouse Assay, Expressing, Ex Vivo, Staining, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

    6) Product Images from "Murine B Cell Development and Antibody Responses to Model Antigens Are Not Impaired in the Absence of the TNF Receptor GITR"

    Article Title: Murine B Cell Development and Antibody Responses to Model Antigens Are Not Impaired in the Absence of the TNF Receptor GITR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031632

    Absence of GITR expression slightly affects generation or maintenance of mature B cells. Bone marrow and spleen cells from GITR +/+ (129S1 strain, white bars) and GITR −/− (black bars) mice were analyzed ex vivo by flow cytometry to determine the numbers of B cells at different developmental stages. The following B220 + B cell subsets were defined in the bone marrow: pro-B (IgM − CD2 − ), pre-B (IgM − CD2 + ), immature B (IgM + IgD − ), transitional B (IgM high IgD low ) and mature/recirculating B (IgM low IgD high ) cells. The following B220 + B cell subsets were defined in the spleen: transitional T1 (CD93 + IgM + CD23 − ), transitional T2 (CD93 + IgM + CD23 + ), transitional T3 (CD93 + IgM low CD23 + ), follicular (FO, CD1d −/low CD21 + ), and marginal zone (MZ, CD1d high CD21 high ). n = 10 from 5 independent experiments. Error bars represent SD; * p
    Figure Legend Snippet: Absence of GITR expression slightly affects generation or maintenance of mature B cells. Bone marrow and spleen cells from GITR +/+ (129S1 strain, white bars) and GITR −/− (black bars) mice were analyzed ex vivo by flow cytometry to determine the numbers of B cells at different developmental stages. The following B220 + B cell subsets were defined in the bone marrow: pro-B (IgM − CD2 − ), pre-B (IgM − CD2 + ), immature B (IgM + IgD − ), transitional B (IgM high IgD low ) and mature/recirculating B (IgM low IgD high ) cells. The following B220 + B cell subsets were defined in the spleen: transitional T1 (CD93 + IgM + CD23 − ), transitional T2 (CD93 + IgM + CD23 + ), transitional T3 (CD93 + IgM low CD23 + ), follicular (FO, CD1d −/low CD21 + ), and marginal zone (MZ, CD1d high CD21 high ). n = 10 from 5 independent experiments. Error bars represent SD; * p

    Techniques Used: Expressing, Mouse Assay, Ex Vivo, Flow Cytometry, Cytometry

    7) Product Images from "Activation of the MEK-ERK Pathway Is Necessary but Not Sufficient for Breaking Central B Cell Tolerance"

    Article Title: Activation of the MEK-ERK Pathway Is Necessary but Not Sufficient for Breaking Central B Cell Tolerance

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00707

    Constitutive extracellular signal-regulated kinase (ERK) activation does not break the central or peripheral tolerance of autoreactive 3-83Ig + B cells in vivo . (A) Representative histograms and bar graph quantification of phospho-ERK (pERK) levels in ex vivo B220 + B cells from the spleen of autoreactive 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. Cells were treated with pervanadate before pERK staining. (B) Absolute number of B220 + cells in the spleens of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (C) Gating strategy and bar graph quantification of splenic B cell populations from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. B cell subsets were discriminated as: immature/transitional 1 B cells (B220 + CD1d – CD24 hi CD23 – ), transitional 2 B cells (B220 + CD1d – CD24 hi CD23 + ), follicular B cells (B220 + CD1d – CD24 lo CD23 + ), and marginal zone B cells (B220 + CD1d + CD21 hi ). (D) Gating strategy and quantification of IgM + B cell numbers from the spleens of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (E) Gating strategy and quantification of the percentage of λ + cells within B220 + B cells from the spleen of 3-83Igi-H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. For panels (A–E) , N = 9 total 3-83Igi,H-2 b -mb1Cre mice, and N = 7 total 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice, analyzed in four independent experiments. (F) Gating strategy and quantification of T3 B cell numbers (CD23 + IgM lo ) within the transitional B cell population (B220 + CD1d – CD24 hi ) from the spleen of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. In panels (A–F) , B cells from 3-83Igi,H-2 b R26-LSL-caMEK-GFP-mb1Cre mice were additionally gated on GFP + to analyze only the cells expressing caMEK. (G) Concentration (μg/mL) of 3-83IgM and 3-83IgG2a in the sera of 3-83Igi,H-2 b -mb1Cre controls ( N = 7) and 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice ( N = 5). The bottom dotted lines represent background detection levels from a wild-type control and the top dotted lines represent Ig levels present in 3-83,H-2 d nonautoreactive (NA) positive control mice. * P ≤ 0.05; NS, not significant.
    Figure Legend Snippet: Constitutive extracellular signal-regulated kinase (ERK) activation does not break the central or peripheral tolerance of autoreactive 3-83Ig + B cells in vivo . (A) Representative histograms and bar graph quantification of phospho-ERK (pERK) levels in ex vivo B220 + B cells from the spleen of autoreactive 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. Cells were treated with pervanadate before pERK staining. (B) Absolute number of B220 + cells in the spleens of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (C) Gating strategy and bar graph quantification of splenic B cell populations from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. B cell subsets were discriminated as: immature/transitional 1 B cells (B220 + CD1d – CD24 hi CD23 – ), transitional 2 B cells (B220 + CD1d – CD24 hi CD23 + ), follicular B cells (B220 + CD1d – CD24 lo CD23 + ), and marginal zone B cells (B220 + CD1d + CD21 hi ). (D) Gating strategy and quantification of IgM + B cell numbers from the spleens of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (E) Gating strategy and quantification of the percentage of λ + cells within B220 + B cells from the spleen of 3-83Igi-H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. For panels (A–E) , N = 9 total 3-83Igi,H-2 b -mb1Cre mice, and N = 7 total 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice, analyzed in four independent experiments. (F) Gating strategy and quantification of T3 B cell numbers (CD23 + IgM lo ) within the transitional B cell population (B220 + CD1d – CD24 hi ) from the spleen of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. In panels (A–F) , B cells from 3-83Igi,H-2 b R26-LSL-caMEK-GFP-mb1Cre mice were additionally gated on GFP + to analyze only the cells expressing caMEK. (G) Concentration (μg/mL) of 3-83IgM and 3-83IgG2a in the sera of 3-83Igi,H-2 b -mb1Cre controls ( N = 7) and 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice ( N = 5). The bottom dotted lines represent background detection levels from a wild-type control and the top dotted lines represent Ig levels present in 3-83,H-2 d nonautoreactive (NA) positive control mice. * P ≤ 0.05; NS, not significant.

    Techniques Used: Activation Assay, In Vivo, Ex Vivo, Mouse Assay, Staining, Expressing, Concentration Assay, Positive Control

    Extracellular signal-regulated kinase (ERK) activation drives differentiation of BCR-low immature B cells in vitro . (A,B) Representative histograms and bar graph quantification of pERK (A) and BAFFR (B) in bone marrow immature (B220 + IgD – ) B cells cultured for 4 days with IL-7. The cells analyzed were either nonautoreactive (NA) (3-83Igi,H-2 d ) BCR normal cells (NA, black solid line) or BCR-low cells transduced with MIG (black dashed line), caMEK (blue line), or caNRas (red line). The analysis of transduced cells was performed on GFP + cells in all experiments. Staining of pERK was done on cells treated with pervanadate to allow for pERK signal detection. The gray shaded histogram in (A) represents isotype control antibody. (C) Representative histograms and bar graph quantification of the frequency of CD21 + cells in the B220 + B cell population after 3 days of culture with BAFF. (D,E) BCR-low B cells transduced with either caMEK (blue) or MIG control (black) were gated based on increasing GFP expression, which correlates with caMEK expression in caMEK transduced cells. The pERK MFI (D) or the frequency of CD21 + cells (E) were plotted against the GFP MFI of the individual segments. In all panels, N = 3 total, from three independent experiments. * P ≤ 0.05; NS, not significant.
    Figure Legend Snippet: Extracellular signal-regulated kinase (ERK) activation drives differentiation of BCR-low immature B cells in vitro . (A,B) Representative histograms and bar graph quantification of pERK (A) and BAFFR (B) in bone marrow immature (B220 + IgD – ) B cells cultured for 4 days with IL-7. The cells analyzed were either nonautoreactive (NA) (3-83Igi,H-2 d ) BCR normal cells (NA, black solid line) or BCR-low cells transduced with MIG (black dashed line), caMEK (blue line), or caNRas (red line). The analysis of transduced cells was performed on GFP + cells in all experiments. Staining of pERK was done on cells treated with pervanadate to allow for pERK signal detection. The gray shaded histogram in (A) represents isotype control antibody. (C) Representative histograms and bar graph quantification of the frequency of CD21 + cells in the B220 + B cell population after 3 days of culture with BAFF. (D,E) BCR-low B cells transduced with either caMEK (blue) or MIG control (black) were gated based on increasing GFP expression, which correlates with caMEK expression in caMEK transduced cells. The pERK MFI (D) or the frequency of CD21 + cells (E) were plotted against the GFP MFI of the individual segments. In all panels, N = 3 total, from three independent experiments. * P ≤ 0.05; NS, not significant.

    Techniques Used: Activation Assay, In Vitro, Cell Culture, Transduction, Staining, Expressing

    Extracellular signal-regulated kinase (ERK) activation drives the differentiation of low-avidity, but not high-avidity autoreactive B cells in vitro . (A,B) Representative histograms and bar graph quantification of phospho-ERK (pERK) (A) and BAFFR (B) in bone marrow immature B cells (B220 + IgD – ) cultured for 4 days with IL-7. The B cells analyzed were either non-transduced nonautoreactive (NA) (3-83Igi,H-2 d ) cells (black solid line) or autoreactive (3-83Igi,H-2 b ) cells transduced with MIG control (black dashed line), caMEK (blue line), or caNRas (red line) retroviral vectors. Transduced cells were gated on GFP + for analyses. Staining of pERK (in A) was done on cells treated with pervanadate. The gray shaded histogram in (A) represents isotype control antibody. (C) Representative histograms and bar graph quantification of the frequency of CD21 + cells in the B220 + B cell population after 3 days of culture with BAFF. (D) Schematic of the system utilized to investigate the effect of caMEK on the in vitro differentiation of immature B cells cultured with increasing amounts of BCR stimulation. 3-83Igi,H-2 d bone marrow cells were cultured with IL-7 for 4 days and transduced with retrovirus carrying either caMEK or MIG on the second day of culture. Cells were then incubated with BAFF and increasing amounts of an agonistic anti-3-83Ig idiotypic mAb (S23), this latter added each day during a 3 days culture. (E) CD21 expression (MFI) on B cells treated as described in (D) : caMEK-GFP, blue bars and MIG, white bars. In all panels, N = 3 total, from three independent experiments. * P ≤ 0.05; NS, not significant.
    Figure Legend Snippet: Extracellular signal-regulated kinase (ERK) activation drives the differentiation of low-avidity, but not high-avidity autoreactive B cells in vitro . (A,B) Representative histograms and bar graph quantification of phospho-ERK (pERK) (A) and BAFFR (B) in bone marrow immature B cells (B220 + IgD – ) cultured for 4 days with IL-7. The B cells analyzed were either non-transduced nonautoreactive (NA) (3-83Igi,H-2 d ) cells (black solid line) or autoreactive (3-83Igi,H-2 b ) cells transduced with MIG control (black dashed line), caMEK (blue line), or caNRas (red line) retroviral vectors. Transduced cells were gated on GFP + for analyses. Staining of pERK (in A) was done on cells treated with pervanadate. The gray shaded histogram in (A) represents isotype control antibody. (C) Representative histograms and bar graph quantification of the frequency of CD21 + cells in the B220 + B cell population after 3 days of culture with BAFF. (D) Schematic of the system utilized to investigate the effect of caMEK on the in vitro differentiation of immature B cells cultured with increasing amounts of BCR stimulation. 3-83Igi,H-2 d bone marrow cells were cultured with IL-7 for 4 days and transduced with retrovirus carrying either caMEK or MIG on the second day of culture. Cells were then incubated with BAFF and increasing amounts of an agonistic anti-3-83Ig idiotypic mAb (S23), this latter added each day during a 3 days culture. (E) CD21 expression (MFI) on B cells treated as described in (D) : caMEK-GFP, blue bars and MIG, white bars. In all panels, N = 3 total, from three independent experiments. * P ≤ 0.05; NS, not significant.

    Techniques Used: Activation Assay, In Vitro, Cell Culture, Transduction, Staining, Incubation, Expressing

    Constitutive extracellular signal-regulated kinase (ERK) activation does not rescue the in vivo development of autoreactive bone marrow B cells. (A) Schematic of the in vivo model for caMEK expression in autoreactive B cells. (B) Representative histograms and bar graph quantification of pERK levels in ex vivo immature B cells (B220 + IgD – ) from the bone marrow of NA (3-83Igi,H-2 d ) mice and autoreactive (3-83Igi,H-2 b ) mb1Cre or R26-LSL-caMEK-GFP-mb1Cre (caMEK-mb1Cre) mice. Cells were treated with pervanadate before pERK staining. (C) Absolute numbers of B220 + cells in the bone marrow of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (D) Gating strategy and bar graph quantification of cell numbers in bone marrow B cell populations from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. B cell subsets were discriminated as: immature B cells (B220 + CD24 hi CD21 – CD23 – ), T1-like B cells (B220 + CD24 hi CD21 + CD23 – ), T2-like B cells (B220 + CD24 hi CD21 ± CD23 + ), and recirculating mature B cells (B220 + CD24 lo CD21 hi ). (E) Mean fluorescence intensity of IgM surface expression on B cells belonging to the B cell subsets gated as in (D) . (F) Gating strategy and quantification of the number of IgM + (edited) and IgM – (editing) cells within the bone marrow immature (B220 + CD24 hi ) B cell population from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (G) Gating strategy and quantification of the percentage of λ+ cells within immature (B220 + CD24 hi ) bone marrow B cells from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. For panels (A–F) , N = 9 total 3-83Igi,H-2 b -mb1Cre mice and N = 7 total 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice, analyzed in four independent experiments. (H) Relative Rag1 and Rag2 mRNA levels in B220 + or B220 + GFP + cells sorted from the bone marrow of 3-83Igi,H-2 b -mb1Cre or 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice, respectively. Data were normalized to 18 s mRNA levels and are expressed as fold change over the average mRNA levels in 3-83Igi,H-2 b -mb1Cre cells. N = 3 mice from one experiment. In all panels, B cells from 3-83Igi,H-2 b R26-LSL-caMEK-GFP-mb1Cre mice were additionally gated on GFP + to analyze only the cells expressing caMEK. ** P ≤ 0.01, *** P ≤ 0.001, NS, not significant.
    Figure Legend Snippet: Constitutive extracellular signal-regulated kinase (ERK) activation does not rescue the in vivo development of autoreactive bone marrow B cells. (A) Schematic of the in vivo model for caMEK expression in autoreactive B cells. (B) Representative histograms and bar graph quantification of pERK levels in ex vivo immature B cells (B220 + IgD – ) from the bone marrow of NA (3-83Igi,H-2 d ) mice and autoreactive (3-83Igi,H-2 b ) mb1Cre or R26-LSL-caMEK-GFP-mb1Cre (caMEK-mb1Cre) mice. Cells were treated with pervanadate before pERK staining. (C) Absolute numbers of B220 + cells in the bone marrow of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (D) Gating strategy and bar graph quantification of cell numbers in bone marrow B cell populations from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. B cell subsets were discriminated as: immature B cells (B220 + CD24 hi CD21 – CD23 – ), T1-like B cells (B220 + CD24 hi CD21 + CD23 – ), T2-like B cells (B220 + CD24 hi CD21 ± CD23 + ), and recirculating mature B cells (B220 + CD24 lo CD21 hi ). (E) Mean fluorescence intensity of IgM surface expression on B cells belonging to the B cell subsets gated as in (D) . (F) Gating strategy and quantification of the number of IgM + (edited) and IgM – (editing) cells within the bone marrow immature (B220 + CD24 hi ) B cell population from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (G) Gating strategy and quantification of the percentage of λ+ cells within immature (B220 + CD24 hi ) bone marrow B cells from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. For panels (A–F) , N = 9 total 3-83Igi,H-2 b -mb1Cre mice and N = 7 total 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice, analyzed in four independent experiments. (H) Relative Rag1 and Rag2 mRNA levels in B220 + or B220 + GFP + cells sorted from the bone marrow of 3-83Igi,H-2 b -mb1Cre or 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice, respectively. Data were normalized to 18 s mRNA levels and are expressed as fold change over the average mRNA levels in 3-83Igi,H-2 b -mb1Cre cells. N = 3 mice from one experiment. In all panels, B cells from 3-83Igi,H-2 b R26-LSL-caMEK-GFP-mb1Cre mice were additionally gated on GFP + to analyze only the cells expressing caMEK. ** P ≤ 0.01, *** P ≤ 0.001, NS, not significant.

    Techniques Used: Activation Assay, In Vivo, Expressing, Ex Vivo, Mouse Assay, Staining, Fluorescence

    8) Product Images from "Lack of T cells in Act1-deficient mice results in elevated IgM-specific autoantibodies but reduced lupus-like disease"

    Article Title: Lack of T cells in Act1-deficient mice results in elevated IgM-specific autoantibodies but reduced lupus-like disease

    Journal: European journal of immunology

    doi: 10.1002/eji.201142238

    B6.Act1 −/− as well as TCRβ/δ-deficient mice develop significantly increased levels of MZ B cells. Splenic MZ B cells (B220 + CD21 high CD23 low IgM high ) were identified in 16-18 week old WT (n = 7), TCRβ/δ −/−
    Figure Legend Snippet: B6.Act1 −/− as well as TCRβ/δ-deficient mice develop significantly increased levels of MZ B cells. Splenic MZ B cells (B220 + CD21 high CD23 low IgM high ) were identified in 16-18 week old WT (n = 7), TCRβ/δ −/−

    Techniques Used: Mouse Assay

    9) Product Images from "Defective lymphoid organogenesis underlies the immune deficiency caused by a heterozygous S32I mutation in IκBα"

    Article Title: Defective lymphoid organogenesis underlies the immune deficiency caused by a heterozygous S32I mutation in IκBα

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20140979

    Secondary lymphoid organs and immune function in WT→IκBα mutant chimeras. (A) Photograph of inguinal fat pad with the vessel junction. The dotted circle indicates the LN at the vessel junction in the fat pad. (B) H E staining of small intestine sections. The arrow points to a PP in the WT mouse. (C) Spleen sections were stained with H E (left) and by immunofluorescence for FDCM1, IgD, and IgM (right). (D) Percentage of CD21 + CD23 − MZ B cells among B220 + cells in the spleen, as determined by flow cytometry ( n = 4–6 mice per group). (E–G) Increase in ear thickness measured by a micrometer (E), representative H E-stained sections (F), and Ifng and Il-4 mRNA expression determined by qPCR (G) 24 h after ear challenge with OXA or vehicle ( n = 3–5 mice per group). (H and I) Serum TNP-specific (H) and OVA-specific (I) antibody measured by ELISA in mice immunized with TNP-Ficoll (H) and OVA (I), respectively ( n = 5 mice per group). (J) Spleen sections from immunized mice stained by histochemistry with anti-B220 mAb or PNA. Bars: (B) 500 µm; (C [left], F, and J) 200 µm; (C, right) 100 µm. (K) Percentage of PNA + B cells among the B220 + population of splenic B cells determined by flow cytometry ( n = 3–5 mice per group). Data in A–C and J are representative of three to six mice. Columns and bars in D, E, G–I, and K represent mean and SD. *, P
    Figure Legend Snippet: Secondary lymphoid organs and immune function in WT→IκBα mutant chimeras. (A) Photograph of inguinal fat pad with the vessel junction. The dotted circle indicates the LN at the vessel junction in the fat pad. (B) H E staining of small intestine sections. The arrow points to a PP in the WT mouse. (C) Spleen sections were stained with H E (left) and by immunofluorescence for FDCM1, IgD, and IgM (right). (D) Percentage of CD21 + CD23 − MZ B cells among B220 + cells in the spleen, as determined by flow cytometry ( n = 4–6 mice per group). (E–G) Increase in ear thickness measured by a micrometer (E), representative H E-stained sections (F), and Ifng and Il-4 mRNA expression determined by qPCR (G) 24 h after ear challenge with OXA or vehicle ( n = 3–5 mice per group). (H and I) Serum TNP-specific (H) and OVA-specific (I) antibody measured by ELISA in mice immunized with TNP-Ficoll (H) and OVA (I), respectively ( n = 5 mice per group). (J) Spleen sections from immunized mice stained by histochemistry with anti-B220 mAb or PNA. Bars: (B) 500 µm; (C [left], F, and J) 200 µm; (C, right) 100 µm. (K) Percentage of PNA + B cells among the B220 + population of splenic B cells determined by flow cytometry ( n = 3–5 mice per group). Data in A–C and J are representative of three to six mice. Columns and bars in D, E, G–I, and K represent mean and SD. *, P

    Techniques Used: Mutagenesis, Staining, Immunofluorescence, Flow Cytometry, Cytometry, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Defective lymphoid organogenesis in IκBα mutant→ Rag2 −/− chimeras. (A) Inguinal LN sections stained with H E. (B) Numbers of total cells and B and T cells from inguinal and axillary LNs (one from each mouse) evaluated by counting in a hemocytometer. (C) H E staining of longitudinal sections of the small intestine. (D) Spleen sections were stained with H E (left) and by immunofluorescence for B220 and FDCM1 (right). Bars: (A and D [right]) 100 µm; (C) 1,000 µm; (D, left) 200 µm. (E) Percentage of CD21 + CD23 − MZ B cells among the B220 + cells in the spleen determined by flow cytometry. Data in A–E are representative of four to six mice per group in three to four independent experiments. Columns and bars in B and E represent mean and SD. **, P
    Figure Legend Snippet: Defective lymphoid organogenesis in IκBα mutant→ Rag2 −/− chimeras. (A) Inguinal LN sections stained with H E. (B) Numbers of total cells and B and T cells from inguinal and axillary LNs (one from each mouse) evaluated by counting in a hemocytometer. (C) H E staining of longitudinal sections of the small intestine. (D) Spleen sections were stained with H E (left) and by immunofluorescence for B220 and FDCM1 (right). Bars: (A and D [right]) 100 µm; (C) 1,000 µm; (D, left) 200 µm. (E) Percentage of CD21 + CD23 − MZ B cells among the B220 + cells in the spleen determined by flow cytometry. Data in A–E are representative of four to six mice per group in three to four independent experiments. Columns and bars in B and E represent mean and SD. **, P

    Techniques Used: Mutagenesis, Staining, Immunofluorescence, Flow Cytometry, Cytometry, Mouse Assay

    IκBα mutant mice lack splenic follicles, MZs, MZ B cells, and FDCs. (A) Blood lymphocyte numbers in IκBα mutant mice and WT littermates evaluated by CBC analysis using HEMAVET 950FS. Data are representative of three mice per group in two independent experiments. (B–D) Numbers of total splenocytes (B), CD3 + , CD4 + , and CD8 + T cells (C), and B220 + B cells (D) in IκBα mutant mice and WT littermates calculated by multiplying the spleen cell counts evaluated in a hemocytometer by the percentage of lymphocytes and those of CD3 + , CD4 + , and CD8 + T cells and of B220 + obtained by flow cytometry. Data are representative of three to eight mice per group in three independent experiments. (E and F) Distribution of CD93 + transitional (Trans) B cells, CD21 − CD23 + follicular (FO) B cells, and CD21 + CD23 − MZ B cells in gated B220 + splenic B cells (E) and percentages of these cells among splenic B220 + cells (F) in IκBα mutant mice and WT littermates analyzed by flow cytometry. Data are representative of three to six mice per group in three independent experiments. (G–J) Sections of naive spleens stained with H E (G) and the presence of cell staining for MOMA, IgM, and IgD (H), Madcam1 and CD4 (I), and FDCM1 and B220 (J) as determined by immunofluorescence. Bars: (G) 500 µm; (H–J) 100 µm. Data in G–J is representative of a minimum of four mice per group in three independent experiments. Columns and bars represent mean and SD. **, P
    Figure Legend Snippet: IκBα mutant mice lack splenic follicles, MZs, MZ B cells, and FDCs. (A) Blood lymphocyte numbers in IκBα mutant mice and WT littermates evaluated by CBC analysis using HEMAVET 950FS. Data are representative of three mice per group in two independent experiments. (B–D) Numbers of total splenocytes (B), CD3 + , CD4 + , and CD8 + T cells (C), and B220 + B cells (D) in IκBα mutant mice and WT littermates calculated by multiplying the spleen cell counts evaluated in a hemocytometer by the percentage of lymphocytes and those of CD3 + , CD4 + , and CD8 + T cells and of B220 + obtained by flow cytometry. Data are representative of three to eight mice per group in three independent experiments. (E and F) Distribution of CD93 + transitional (Trans) B cells, CD21 − CD23 + follicular (FO) B cells, and CD21 + CD23 − MZ B cells in gated B220 + splenic B cells (E) and percentages of these cells among splenic B220 + cells (F) in IκBα mutant mice and WT littermates analyzed by flow cytometry. Data are representative of three to six mice per group in three independent experiments. (G–J) Sections of naive spleens stained with H E (G) and the presence of cell staining for MOMA, IgM, and IgD (H), Madcam1 and CD4 (I), and FDCM1 and B220 (J) as determined by immunofluorescence. Bars: (G) 500 µm; (H–J) 100 µm. Data in G–J is representative of a minimum of four mice per group in three independent experiments. Columns and bars represent mean and SD. **, P

    Techniques Used: Mutagenesis, Mouse Assay, Flow Cytometry, Cytometry, Staining, Immunofluorescence

    10) Product Images from "An Essential Role for RGS Protein/Gαi2 Interactions in B Lymphocyte Directed Cell Migration and Trafficking"

    Article Title: An Essential Role for RGS Protein/Gαi2 Interactions in B Lymphocyte Directed Cell Migration and Trafficking

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

    doi: 10.4049/jimmunol.1401952

    Small LN follicles and spleen B cell follicles in the G184S KI mice. (A) Confocal microscopy of the inguinal LN of a wild type and a G184S KI bone marrow reconstituted mice. LNs were fixed and immunostained with directly fluorescently labeled antibodies recognizing B220 (green), CD4 (blue), Ki67 (pink), CD169 (red), and CD21/35 (turquoise). The images were tiled to reconstruct an image of the entire LN. Shown below are 3× electronic magnification focused on one of the LN follicles (F) and the adjacent T cell zone (TCZ). Boxed area shows numerous G184S KI B cells located in the TCZ. Scale bars are 300 µm (top) and 100 µm (bottom). (B) Confocal microscopy of the spleens from a wild type and a G184S KI bone marrow reconstituted mice. A thick splenic section was fixed and immunostained with directly fluorescently labeled antibodies recognizing B220 (white), CD4 (blue), Ki67 (pink), CD169 (red), and CD21/35 (green). The images were tiled to reconstruct an image of the entire spleen. GCs are indicated with white arrowheads. Shown below are 4× electronic magnification focused on the splenic white pulp. The splenic B cell follicle (F) and T cell zone (TCZ) are indicated. Scale bars are 400 µm (top) and 100 µm (bottom). (C D) Confocal microscopy of spleens from a wild type and a G184S KI bone marrow reconstituted mice immunized 10 days previously with sheep RBCs. A thick splenic section was fixed and immunostained with directly fluorescently labeled antibodies recognizing B220 (white), CD4 (blue), Ki67 (pink), CD169 (red), and CD21/35 (green). The images were tiled to reconstruct an image of the entire spleen (C). GCs are indicated with white arrowheads. Scale bars are 400 um. Electronic zoomed images (D) are shown on the right. In the bottom images the B220 signal was removed to allow better visualization of the GC structure. The T cell zone (TCZ) is indicated. Scale bars are 100 µm.
    Figure Legend Snippet: Small LN follicles and spleen B cell follicles in the G184S KI mice. (A) Confocal microscopy of the inguinal LN of a wild type and a G184S KI bone marrow reconstituted mice. LNs were fixed and immunostained with directly fluorescently labeled antibodies recognizing B220 (green), CD4 (blue), Ki67 (pink), CD169 (red), and CD21/35 (turquoise). The images were tiled to reconstruct an image of the entire LN. Shown below are 3× electronic magnification focused on one of the LN follicles (F) and the adjacent T cell zone (TCZ). Boxed area shows numerous G184S KI B cells located in the TCZ. Scale bars are 300 µm (top) and 100 µm (bottom). (B) Confocal microscopy of the spleens from a wild type and a G184S KI bone marrow reconstituted mice. A thick splenic section was fixed and immunostained with directly fluorescently labeled antibodies recognizing B220 (white), CD4 (blue), Ki67 (pink), CD169 (red), and CD21/35 (green). The images were tiled to reconstruct an image of the entire spleen. GCs are indicated with white arrowheads. Shown below are 4× electronic magnification focused on the splenic white pulp. The splenic B cell follicle (F) and T cell zone (TCZ) are indicated. Scale bars are 400 µm (top) and 100 µm (bottom). (C D) Confocal microscopy of spleens from a wild type and a G184S KI bone marrow reconstituted mice immunized 10 days previously with sheep RBCs. A thick splenic section was fixed and immunostained with directly fluorescently labeled antibodies recognizing B220 (white), CD4 (blue), Ki67 (pink), CD169 (red), and CD21/35 (green). The images were tiled to reconstruct an image of the entire spleen (C). GCs are indicated with white arrowheads. Scale bars are 400 um. Electronic zoomed images (D) are shown on the right. In the bottom images the B220 signal was removed to allow better visualization of the GC structure. The T cell zone (TCZ) is indicated. Scale bars are 100 µm.

    Techniques Used: Mouse Assay, Confocal Microscopy, Labeling

    11) Product Images from "Activation of the MEK-ERK Pathway Is Necessary but Not Sufficient for Breaking Central B Cell Tolerance"

    Article Title: Activation of the MEK-ERK Pathway Is Necessary but Not Sufficient for Breaking Central B Cell Tolerance

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00707

    Constitutive extracellular signal-regulated kinase (ERK) activation does not break the central or peripheral tolerance of autoreactive 3-83Ig + B cells in vivo . (A) Representative histograms and bar graph quantification of phospho-ERK (pERK) levels in ex vivo B220 + B cells from the spleen of autoreactive 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. Cells were treated with pervanadate before pERK staining. (B) Absolute number of B220 + cells in the spleens of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (C) Gating strategy and bar graph quantification of splenic B cell populations from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. B cell subsets were discriminated as: immature/transitional 1 B cells (B220 + CD1d – CD24 hi CD23 – ), transitional 2 B cells (B220 + CD1d – CD24 hi CD23 + ), follicular B cells (B220 + CD1d – CD24 lo CD23 + ), and marginal zone B cells (B220 + CD1d + CD21 hi ). (D) Gating strategy and quantification of IgM + B cell numbers from the spleens of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (E) Gating strategy and quantification of the percentage of λ + cells within B220 + B cells from the spleen of 3-83Igi-H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. For panels (A–E) , N = 9 total 3-83Igi,H-2 b -mb1Cre mice, and N = 7 total 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice, analyzed in four independent experiments. (F) Gating strategy and quantification of T3 B cell numbers (CD23 + IgM lo ) within the transitional B cell population (B220 + CD1d – CD24 hi ) from the spleen of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. In panels (A–F) , B cells from 3-83Igi,H-2 b R26-LSL-caMEK-GFP-mb1Cre mice were additionally gated on GFP + to analyze only the cells expressing caMEK. (G) Concentration (μg/mL) of 3-83IgM and 3-83IgG2a in the sera of 3-83Igi,H-2 b -mb1Cre controls ( N = 7) and 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice ( N = 5). The bottom dotted lines represent background detection levels from a wild-type control and the top dotted lines represent Ig levels present in 3-83,H-2 d nonautoreactive (NA) positive control mice. * P ≤ 0.05; NS, not significant.
    Figure Legend Snippet: Constitutive extracellular signal-regulated kinase (ERK) activation does not break the central or peripheral tolerance of autoreactive 3-83Ig + B cells in vivo . (A) Representative histograms and bar graph quantification of phospho-ERK (pERK) levels in ex vivo B220 + B cells from the spleen of autoreactive 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. Cells were treated with pervanadate before pERK staining. (B) Absolute number of B220 + cells in the spleens of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (C) Gating strategy and bar graph quantification of splenic B cell populations from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. B cell subsets were discriminated as: immature/transitional 1 B cells (B220 + CD1d – CD24 hi CD23 – ), transitional 2 B cells (B220 + CD1d – CD24 hi CD23 + ), follicular B cells (B220 + CD1d – CD24 lo CD23 + ), and marginal zone B cells (B220 + CD1d + CD21 hi ). (D) Gating strategy and quantification of IgM + B cell numbers from the spleens of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (E) Gating strategy and quantification of the percentage of λ + cells within B220 + B cells from the spleen of 3-83Igi-H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. For panels (A–E) , N = 9 total 3-83Igi,H-2 b -mb1Cre mice, and N = 7 total 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice, analyzed in four independent experiments. (F) Gating strategy and quantification of T3 B cell numbers (CD23 + IgM lo ) within the transitional B cell population (B220 + CD1d – CD24 hi ) from the spleen of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. In panels (A–F) , B cells from 3-83Igi,H-2 b R26-LSL-caMEK-GFP-mb1Cre mice were additionally gated on GFP + to analyze only the cells expressing caMEK. (G) Concentration (μg/mL) of 3-83IgM and 3-83IgG2a in the sera of 3-83Igi,H-2 b -mb1Cre controls ( N = 7) and 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice ( N = 5). The bottom dotted lines represent background detection levels from a wild-type control and the top dotted lines represent Ig levels present in 3-83,H-2 d nonautoreactive (NA) positive control mice. * P ≤ 0.05; NS, not significant.

    Techniques Used: Activation Assay, In Vivo, Ex Vivo, Mouse Assay, Staining, Expressing, Concentration Assay, Positive Control

    Extracellular signal-regulated kinase (ERK) activation drives differentiation of BCR-low immature B cells in vitro . (A,B) Representative histograms and bar graph quantification of pERK (A) and BAFFR (B) in bone marrow immature (B220 + IgD – ) B cells cultured for 4 days with IL-7. The cells analyzed were either nonautoreactive (NA) (3-83Igi,H-2 d ) BCR normal cells (NA, black solid line) or BCR-low cells transduced with MIG (black dashed line), caMEK (blue line), or caNRas (red line). The analysis of transduced cells was performed on GFP + cells in all experiments. Staining of pERK was done on cells treated with pervanadate to allow for pERK signal detection. The gray shaded histogram in (A) represents isotype control antibody. (C) Representative histograms and bar graph quantification of the frequency of CD21 + cells in the B220 + B cell population after 3 days of culture with BAFF. (D,E) BCR-low B cells transduced with either caMEK (blue) or MIG control (black) were gated based on increasing GFP expression, which correlates with caMEK expression in caMEK transduced cells. The pERK MFI (D) or the frequency of CD21 + cells (E) were plotted against the GFP MFI of the individual segments. In all panels, N = 3 total, from three independent experiments. * P ≤ 0.05; NS, not significant.
    Figure Legend Snippet: Extracellular signal-regulated kinase (ERK) activation drives differentiation of BCR-low immature B cells in vitro . (A,B) Representative histograms and bar graph quantification of pERK (A) and BAFFR (B) in bone marrow immature (B220 + IgD – ) B cells cultured for 4 days with IL-7. The cells analyzed were either nonautoreactive (NA) (3-83Igi,H-2 d ) BCR normal cells (NA, black solid line) or BCR-low cells transduced with MIG (black dashed line), caMEK (blue line), or caNRas (red line). The analysis of transduced cells was performed on GFP + cells in all experiments. Staining of pERK was done on cells treated with pervanadate to allow for pERK signal detection. The gray shaded histogram in (A) represents isotype control antibody. (C) Representative histograms and bar graph quantification of the frequency of CD21 + cells in the B220 + B cell population after 3 days of culture with BAFF. (D,E) BCR-low B cells transduced with either caMEK (blue) or MIG control (black) were gated based on increasing GFP expression, which correlates with caMEK expression in caMEK transduced cells. The pERK MFI (D) or the frequency of CD21 + cells (E) were plotted against the GFP MFI of the individual segments. In all panels, N = 3 total, from three independent experiments. * P ≤ 0.05; NS, not significant.

    Techniques Used: Activation Assay, In Vitro, Cell Culture, Transduction, Staining, Expressing

    Extracellular signal-regulated kinase (ERK) activation drives the differentiation of low-avidity, but not high-avidity autoreactive B cells in vitro . (A,B) Representative histograms and bar graph quantification of phospho-ERK (pERK) (A) and BAFFR (B) in bone marrow immature B cells (B220 + IgD – ) cultured for 4 days with IL-7. The B cells analyzed were either non-transduced nonautoreactive (NA) (3-83Igi,H-2 d ) cells (black solid line) or autoreactive (3-83Igi,H-2 b ) cells transduced with MIG control (black dashed line), caMEK (blue line), or caNRas (red line) retroviral vectors. Transduced cells were gated on GFP + for analyses. Staining of pERK (in A) was done on cells treated with pervanadate. The gray shaded histogram in (A) represents isotype control antibody. (C) Representative histograms and bar graph quantification of the frequency of CD21 + cells in the B220 + B cell population after 3 days of culture with BAFF. (D) Schematic of the system utilized to investigate the effect of caMEK on the in vitro differentiation of immature B cells cultured with increasing amounts of BCR stimulation. 3-83Igi,H-2 d bone marrow cells were cultured with IL-7 for 4 days and transduced with retrovirus carrying either caMEK or MIG on the second day of culture. Cells were then incubated with BAFF and increasing amounts of an agonistic anti-3-83Ig idiotypic mAb (S23), this latter added each day during a 3 days culture. (E) CD21 expression (MFI) on B cells treated as described in (D) : caMEK-GFP, blue bars and MIG, white bars. In all panels, N = 3 total, from three independent experiments. * P ≤ 0.05; NS, not significant.
    Figure Legend Snippet: Extracellular signal-regulated kinase (ERK) activation drives the differentiation of low-avidity, but not high-avidity autoreactive B cells in vitro . (A,B) Representative histograms and bar graph quantification of phospho-ERK (pERK) (A) and BAFFR (B) in bone marrow immature B cells (B220 + IgD – ) cultured for 4 days with IL-7. The B cells analyzed were either non-transduced nonautoreactive (NA) (3-83Igi,H-2 d ) cells (black solid line) or autoreactive (3-83Igi,H-2 b ) cells transduced with MIG control (black dashed line), caMEK (blue line), or caNRas (red line) retroviral vectors. Transduced cells were gated on GFP + for analyses. Staining of pERK (in A) was done on cells treated with pervanadate. The gray shaded histogram in (A) represents isotype control antibody. (C) Representative histograms and bar graph quantification of the frequency of CD21 + cells in the B220 + B cell population after 3 days of culture with BAFF. (D) Schematic of the system utilized to investigate the effect of caMEK on the in vitro differentiation of immature B cells cultured with increasing amounts of BCR stimulation. 3-83Igi,H-2 d bone marrow cells were cultured with IL-7 for 4 days and transduced with retrovirus carrying either caMEK or MIG on the second day of culture. Cells were then incubated with BAFF and increasing amounts of an agonistic anti-3-83Ig idiotypic mAb (S23), this latter added each day during a 3 days culture. (E) CD21 expression (MFI) on B cells treated as described in (D) : caMEK-GFP, blue bars and MIG, white bars. In all panels, N = 3 total, from three independent experiments. * P ≤ 0.05; NS, not significant.

    Techniques Used: Activation Assay, In Vitro, Cell Culture, Transduction, Staining, Incubation, Expressing

    Constitutive extracellular signal-regulated kinase (ERK) activation does not rescue the in vivo development of autoreactive bone marrow B cells. (A) Schematic of the in vivo model for caMEK expression in autoreactive B cells. (B) Representative histograms and bar graph quantification of pERK levels in ex vivo immature B cells (B220 + IgD – ) from the bone marrow of NA (3-83Igi,H-2 d ) mice and autoreactive (3-83Igi,H-2 b ) mb1Cre or R26-LSL-caMEK-GFP-mb1Cre (caMEK-mb1Cre) mice. Cells were treated with pervanadate before pERK staining. (C) Absolute numbers of B220 + cells in the bone marrow of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (D) Gating strategy and bar graph quantification of cell numbers in bone marrow B cell populations from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. B cell subsets were discriminated as: immature B cells (B220 + CD24 hi CD21 – CD23 – ), T1-like B cells (B220 + CD24 hi CD21 + CD23 – ), T2-like B cells (B220 + CD24 hi CD21 ± CD23 + ), and recirculating mature B cells (B220 + CD24 lo CD21 hi ). (E) Mean fluorescence intensity of IgM surface expression on B cells belonging to the B cell subsets gated as in (D) . (F) Gating strategy and quantification of the number of IgM + (edited) and IgM – (editing) cells within the bone marrow immature (B220 + CD24 hi ) B cell population from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (G) Gating strategy and quantification of the percentage of λ+ cells within immature (B220 + CD24 hi ) bone marrow B cells from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. For panels (A–F) , N = 9 total 3-83Igi,H-2 b -mb1Cre mice and N = 7 total 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice, analyzed in four independent experiments. (H) Relative Rag1 and Rag2 mRNA levels in B220 + or B220 + GFP + cells sorted from the bone marrow of 3-83Igi,H-2 b -mb1Cre or 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice, respectively. Data were normalized to 18 s mRNA levels and are expressed as fold change over the average mRNA levels in 3-83Igi,H-2 b -mb1Cre cells. N = 3 mice from one experiment. In all panels, B cells from 3-83Igi,H-2 b R26-LSL-caMEK-GFP-mb1Cre mice were additionally gated on GFP + to analyze only the cells expressing caMEK. ** P ≤ 0.01, *** P ≤ 0.001, NS, not significant.
    Figure Legend Snippet: Constitutive extracellular signal-regulated kinase (ERK) activation does not rescue the in vivo development of autoreactive bone marrow B cells. (A) Schematic of the in vivo model for caMEK expression in autoreactive B cells. (B) Representative histograms and bar graph quantification of pERK levels in ex vivo immature B cells (B220 + IgD – ) from the bone marrow of NA (3-83Igi,H-2 d ) mice and autoreactive (3-83Igi,H-2 b ) mb1Cre or R26-LSL-caMEK-GFP-mb1Cre (caMEK-mb1Cre) mice. Cells were treated with pervanadate before pERK staining. (C) Absolute numbers of B220 + cells in the bone marrow of 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (D) Gating strategy and bar graph quantification of cell numbers in bone marrow B cell populations from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. B cell subsets were discriminated as: immature B cells (B220 + CD24 hi CD21 – CD23 – ), T1-like B cells (B220 + CD24 hi CD21 + CD23 – ), T2-like B cells (B220 + CD24 hi CD21 ± CD23 + ), and recirculating mature B cells (B220 + CD24 lo CD21 hi ). (E) Mean fluorescence intensity of IgM surface expression on B cells belonging to the B cell subsets gated as in (D) . (F) Gating strategy and quantification of the number of IgM + (edited) and IgM – (editing) cells within the bone marrow immature (B220 + CD24 hi ) B cell population from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. (G) Gating strategy and quantification of the percentage of λ+ cells within immature (B220 + CD24 hi ) bone marrow B cells from 3-83Igi,H-2 b mb1Cre and R26-LSL-caMEK-GFP-mb1Cre mice. For panels (A–F) , N = 9 total 3-83Igi,H-2 b -mb1Cre mice and N = 7 total 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice, analyzed in four independent experiments. (H) Relative Rag1 and Rag2 mRNA levels in B220 + or B220 + GFP + cells sorted from the bone marrow of 3-83Igi,H-2 b -mb1Cre or 3-83Igi,H-2 b -R26-LSL-caMEK-GFP-mb1Cre mice, respectively. Data were normalized to 18 s mRNA levels and are expressed as fold change over the average mRNA levels in 3-83Igi,H-2 b -mb1Cre cells. N = 3 mice from one experiment. In all panels, B cells from 3-83Igi,H-2 b R26-LSL-caMEK-GFP-mb1Cre mice were additionally gated on GFP + to analyze only the cells expressing caMEK. ** P ≤ 0.01, *** P ≤ 0.001, NS, not significant.

    Techniques Used: Activation Assay, In Vivo, Expressing, Ex Vivo, Mouse Assay, Staining, Fluorescence

    12) Product Images from "Impaired Generation of Hepatitis B Virus-specific Memory B Cells in HIV Infected Individuals Following Vaccination"

    Article Title: Impaired Generation of Hepatitis B Virus-specific Memory B Cells in HIV Infected Individuals Following Vaccination

    Journal: Vaccine

    doi: 10.1016/j.vaccine.2010.03.022

    Percentages of circulating (A) CD19+ B cells and (B) CD19+CD27+ memory B cells that are CD21− in HIV uninfected and HIV infected donors and study subjects at entry, week 28 and week 72/76 Open diamonds represent aviremic HIV infected study subjects (plasma HIV-1 RNA
    Figure Legend Snippet: Percentages of circulating (A) CD19+ B cells and (B) CD19+CD27+ memory B cells that are CD21− in HIV uninfected and HIV infected donors and study subjects at entry, week 28 and week 72/76 Open diamonds represent aviremic HIV infected study subjects (plasma HIV-1 RNA

    Techniques Used: Infection

    13) Product Images from "Follicular Dendritic Cells Specifically Express the Long CR2/CD21 Isoform"

    Article Title: Follicular Dendritic Cells Specifically Express the Long CR2/CD21 Isoform

    Journal: The Journal of Experimental Medicine

    doi:

    mAb 7D6 specifically stains FDC networks within tonsillar and splenic GCs or FDCs in isolated form. ( A ) Red mAb 7D6 staining of FDC networks within a GC of human tonsil ( DZ , dark zone; LZ , light zone; ×200). ( B ) Red antiCD21 staining of FDC networks and B lymphocytes within the follicular mantle ( FM ) and extrafollicular area ( A and B show the same secondary follicle on two serial sections). ( C ) mAb 7D6 staining of FDC networks within a primary follicle of human spleen ( CA , central arteriole; ×200). ( D ) anti-CD21 staining of FDC networks as well as follicular B cells and marginal zone ( MZ ) B cells ( C and D show the same splenic white pulp on two serial sections). ( E ) Negative mAb 7D6 staining on human thymus (×200). ( F ) Positive staining of anti-ICAM1/ CD54 on human thymus ( E and F show the same thymic area on two serial sections) ( C , cortex. M , medullar). ( G ) Giemsa staining of isolated FDC-lymphocyte clusters. FDC can be recognized as cells containing one or two big round nuclei with decondensed chromatin and clear nucleoli (×1000). ( H ) mAb 7D6 staining of isolated FDCs.
    Figure Legend Snippet: mAb 7D6 specifically stains FDC networks within tonsillar and splenic GCs or FDCs in isolated form. ( A ) Red mAb 7D6 staining of FDC networks within a GC of human tonsil ( DZ , dark zone; LZ , light zone; ×200). ( B ) Red antiCD21 staining of FDC networks and B lymphocytes within the follicular mantle ( FM ) and extrafollicular area ( A and B show the same secondary follicle on two serial sections). ( C ) mAb 7D6 staining of FDC networks within a primary follicle of human spleen ( CA , central arteriole; ×200). ( D ) anti-CD21 staining of FDC networks as well as follicular B cells and marginal zone ( MZ ) B cells ( C and D show the same splenic white pulp on two serial sections). ( E ) Negative mAb 7D6 staining on human thymus (×200). ( F ) Positive staining of anti-ICAM1/ CD54 on human thymus ( E and F show the same thymic area on two serial sections) ( C , cortex. M , medullar). ( G ) Giemsa staining of isolated FDC-lymphocyte clusters. FDC can be recognized as cells containing one or two big round nuclei with decondensed chromatin and clear nucleoli (×1000). ( H ) mAb 7D6 staining of isolated FDCs.

    Techniques Used: Isolation, Staining

    Isolation of highly purified FDC by FACS ® sorting. ( A ) Low density tonsillar cells were stained by anti–CD21-PE and anti–CD14FITC (detailed in Materials and Methods). FDCs were sorted according to their CD21 ++ CD14 + phenotype. B cells and monocytes could be recognized as CD21 + CD14 − and CD21 − CD14 + cells, respectively. ( B and C ) Giemsa staining of FACS ® sorted FDC (×400 and ×1,000).
    Figure Legend Snippet: Isolation of highly purified FDC by FACS ® sorting. ( A ) Low density tonsillar cells were stained by anti–CD21-PE and anti–CD14FITC (detailed in Materials and Methods). FDCs were sorted according to their CD21 ++ CD14 + phenotype. B cells and monocytes could be recognized as CD21 + CD14 − and CD21 − CD14 + cells, respectively. ( B and C ) Giemsa staining of FACS ® sorted FDC (×400 and ×1,000).

    Techniques Used: Isolation, Purification, FACS, Staining

    Detection of CD21S and CD21L mRNA by PCR among CD21 ++ CD14 + FDCs, IgD + CD38 − FM, and IgD − CD38 + GC B cells. The method for FDC isolation is detailed in Materials and Methods and in Fig. 3 . PCR primers used are indicated in the legend for Fig. 2 . p7D6 cDNA was used as a positive control.
    Figure Legend Snippet: Detection of CD21S and CD21L mRNA by PCR among CD21 ++ CD14 + FDCs, IgD + CD38 − FM, and IgD − CD38 + GC B cells. The method for FDC isolation is detailed in Materials and Methods and in Fig. 3 . PCR primers used are indicated in the legend for Fig. 2 . p7D6 cDNA was used as a positive control.

    Techniques Used: Polymerase Chain Reaction, Isolation, Positive Control

    14) Product Images from "Altered BCR and TLR signals promote enhanced positive selection of autoreactive transitional B cells in Wiskott-Aldrich syndrome"

    Article Title: Altered BCR and TLR signals promote enhanced positive selection of autoreactive transitional B cells in Wiskott-Aldrich syndrome

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20150585

    High-throughput BCR heavy chain sequencing of splenic B cell subsets from WT, Was −/− , and Was fl/fl × Mb-1 cre mice. B cell populations were sorted (total of ∼0.5–10 × 10 6 pooled cells/subset) from B6 WT, Was −/− , and Was fl/fl × Mb-1 cre mice using a minimum of five to six mice/genotype per experiment (three experiments total). RNA was isolated, sequenced, and analyzed using a 5′-RACE 454 platform (see Materials and methods). Data represent a mean of three experiments (15 mice per genotype). (A) Heavy chain variable (VH) gene family usage in bulk WT and Was −/− B cells (∼10 × 10 6 cells/sample). (B) VH gene family usage in sorted WT and Was −/− B220 + CD21 hi CD24 hi CD23 lo CD1d hi MZ B cells (∼0.5–10 6 cells/sample). Additional sequence information is available in Table S1 . Error bars show SEM. Statistical analysis was performed using the Student’s t test: *, P
    Figure Legend Snippet: High-throughput BCR heavy chain sequencing of splenic B cell subsets from WT, Was −/− , and Was fl/fl × Mb-1 cre mice. B cell populations were sorted (total of ∼0.5–10 × 10 6 pooled cells/subset) from B6 WT, Was −/− , and Was fl/fl × Mb-1 cre mice using a minimum of five to six mice/genotype per experiment (three experiments total). RNA was isolated, sequenced, and analyzed using a 5′-RACE 454 platform (see Materials and methods). Data represent a mean of three experiments (15 mice per genotype). (A) Heavy chain variable (VH) gene family usage in bulk WT and Was −/− B cells (∼10 × 10 6 cells/sample). (B) VH gene family usage in sorted WT and Was −/− B220 + CD21 hi CD24 hi CD23 lo CD1d hi MZ B cells (∼0.5–10 6 cells/sample). Additional sequence information is available in Table S1 . Error bars show SEM. Statistical analysis was performed using the Student’s t test: *, P

    Techniques Used: High Throughput Screening Assay, Sequencing, Mouse Assay, Isolation

    Antigen-specific selection of Was −/− transitional B cells requires Myd88 signals. (A) Representative FACS analysis of Ki67 staining of splenic T1 (B220 + CD24 hi CD21 lo ) and T2 (B220 + CD24 hi CD21 mid ) B cells in WT, Was −/− , and Was −/− Myd88 −/− mice. (B) Percentage of Ki67 + B cells in WT ( n = 12), Was −/− ( n = 13), and Was −/− Myd88 −/− ( n = 12) mice. (C) VH gene family usage in sorted WT, Was −/− , and Was −/− Myd88 −/− MZ B cells. Error bars show SEM. Statistical analysis was performed using the Student’s t test: **, P
    Figure Legend Snippet: Antigen-specific selection of Was −/− transitional B cells requires Myd88 signals. (A) Representative FACS analysis of Ki67 staining of splenic T1 (B220 + CD24 hi CD21 lo ) and T2 (B220 + CD24 hi CD21 mid ) B cells in WT, Was −/− , and Was −/− Myd88 −/− mice. (B) Percentage of Ki67 + B cells in WT ( n = 12), Was −/− ( n = 13), and Was −/− Myd88 −/− ( n = 12) mice. (C) VH gene family usage in sorted WT, Was −/− , and Was −/− Myd88 −/− MZ B cells. Error bars show SEM. Statistical analysis was performed using the Student’s t test: **, P

    Techniques Used: Selection, FACS, Staining, Mouse Assay

    Altered specificity of the naive B cell repertoire in WASp-deficient mice. (A) λ-LC usage in splenic B cell populations in 8–10-wk-old B6 ( n = 6), Was fl/fl × Mb-1 cre ( n = 5), and Was −/− ( n = 7) mice assessed by flow cytometry. (B and C) Cloned WT and Was −/− MZ B cell mAb reactivities toward self-antigens (dsDNA, high [PC-4]- and low [PC-14]-affinity phosphorylcholine, MDA-LDL, and Sm-RNP) via ELISA depicted using a pie chart (blue = reactive clones identified based on threshold of 0.5 OD value; gray = nonreactive clones), with percentages of reactive clones and total number of clones tested noted. MZ B cells were FACS sorted and gated based on B220 + CD23 lo CD1d hi CD24 hi CD21 hi surface marker expression from splenocytes pooled from five to six WT or Was −/− mice. (C) ELISA OD values of serial dilution curves of WT and Was −/− MZ mAbs (100 ng/µl). (D) Proportion of low affinity (OD of 0.5–1.5) and high affinity (OD of 1.5–3) in reactive antibody clones to individual self-antigens. (E) Relative binding affinity displayed as AUC of reactive antibodies. (F) Selection of Id (M167) + B cells in peripheral B cell subsets in 10–12-wk-old WT M167 Tg ( n = 9) and Was −/− M167 Tg ( n = 9) mice. Error bars show SEM. Statistical analysis was performed using the Student’s t test: *, P
    Figure Legend Snippet: Altered specificity of the naive B cell repertoire in WASp-deficient mice. (A) λ-LC usage in splenic B cell populations in 8–10-wk-old B6 ( n = 6), Was fl/fl × Mb-1 cre ( n = 5), and Was −/− ( n = 7) mice assessed by flow cytometry. (B and C) Cloned WT and Was −/− MZ B cell mAb reactivities toward self-antigens (dsDNA, high [PC-4]- and low [PC-14]-affinity phosphorylcholine, MDA-LDL, and Sm-RNP) via ELISA depicted using a pie chart (blue = reactive clones identified based on threshold of 0.5 OD value; gray = nonreactive clones), with percentages of reactive clones and total number of clones tested noted. MZ B cells were FACS sorted and gated based on B220 + CD23 lo CD1d hi CD24 hi CD21 hi surface marker expression from splenocytes pooled from five to six WT or Was −/− mice. (C) ELISA OD values of serial dilution curves of WT and Was −/− MZ mAbs (100 ng/µl). (D) Proportion of low affinity (OD of 0.5–1.5) and high affinity (OD of 1.5–3) in reactive antibody clones to individual self-antigens. (E) Relative binding affinity displayed as AUC of reactive antibodies. (F) Selection of Id (M167) + B cells in peripheral B cell subsets in 10–12-wk-old WT M167 Tg ( n = 9) and Was −/− M167 Tg ( n = 9) mice. Error bars show SEM. Statistical analysis was performed using the Student’s t test: *, P

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Clone Assay, Multiple Displacement Amplification, Enzyme-linked Immunosorbent Assay, FACS, Marker, Expressing, Serial Dilution, Binding Assay, Selection

    15) Product Images from "Expression of caveolin by bovine lymphocytes and antigen-presenting cells"

    Article Title: Expression of caveolin by bovine lymphocytes and antigen-presenting cells

    Journal: Immunology

    doi: 10.1046/j.1365-2567.2002.01362.x

    Detection of (a) caveolin-1 and (b) caveolin-3 in bovine immune cells and human and bovine endothelial cells by Western blotting. Caveolin-1 was detected in all cells studied; HEC, human endothelial cell lysate; BAE-1, bovine aortic endothelial cells; MoDC, monocyte-derived dendritic cells; Mo, monocytes; Ma, macrophages; CD4, CD8 and CD21, sorted lymphocyte subsets. The Western blot shows strong labelling of a protein with a molecular mass of ∼22000. The polyclonal antibody against caveolin-3 detected proteins at ∼22000, 44000, 66000 and 210000 in HEC and ∼44000 and 66000 in bovine immune cells.
    Figure Legend Snippet: Detection of (a) caveolin-1 and (b) caveolin-3 in bovine immune cells and human and bovine endothelial cells by Western blotting. Caveolin-1 was detected in all cells studied; HEC, human endothelial cell lysate; BAE-1, bovine aortic endothelial cells; MoDC, monocyte-derived dendritic cells; Mo, monocytes; Ma, macrophages; CD4, CD8 and CD21, sorted lymphocyte subsets. The Western blot shows strong labelling of a protein with a molecular mass of ∼22000. The polyclonal antibody against caveolin-3 detected proteins at ∼22000, 44000, 66000 and 210000 in HEC and ∼44000 and 66000 in bovine immune cells.

    Techniques Used: Western Blot, Derivative Assay

    16) Product Images from "Expression of FcRL4 defines a pro-inflammatory, RANKL-producing B cell subset in rheumatoid arthritis"

    Article Title: Expression of FcRL4 defines a pro-inflammatory, RANKL-producing B cell subset in rheumatoid arthritis

    Journal: Annals of the Rheumatic Diseases

    doi: 10.1136/annrheumdis-2013-204116

    Phenotypic characterisation of FcRL4+ B cells in RA synovial fluid. FcRL4+ and FcRL4− B cells are phenotypically distinct. FcRL4+ B cells have higher expression of CD11c, CD20, CD95, CD80, CD86, CCR1 and CCR5, and lower expression of CD21 compared to FcRL4− B cells. *p
    Figure Legend Snippet: Phenotypic characterisation of FcRL4+ B cells in RA synovial fluid. FcRL4+ and FcRL4− B cells are phenotypically distinct. FcRL4+ B cells have higher expression of CD11c, CD20, CD95, CD80, CD86, CCR1 and CCR5, and lower expression of CD21 compared to FcRL4− B cells. *p

    Techniques Used: Expressing

    17) Product Images from "Human amnion epithelial cells modulate the inflammatory response to ventilation in preterm lambs"

    Article Title: Human amnion epithelial cells modulate the inflammatory response to ventilation in preterm lambs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0173572

    Cell phenotypes in bronchoalveolar lavage fluid samples. CD4 + (A), CD8 + (B), CD21 + (C), γδ + (D), CD25 + (E), CLII + (F) and CD44 + expression of total BAL cells (white bars), lymphocytes (light grey), macrophages (dark grey) and granulocytes (black bars; panel F only) of control (n = 6), vehicle- (n = 5) and hAEC-treated ventilated lambs (n = 6). # p
    Figure Legend Snippet: Cell phenotypes in bronchoalveolar lavage fluid samples. CD4 + (A), CD8 + (B), CD21 + (C), γδ + (D), CD25 + (E), CLII + (F) and CD44 + expression of total BAL cells (white bars), lymphocytes (light grey), macrophages (dark grey) and granulocytes (black bars; panel F only) of control (n = 6), vehicle- (n = 5) and hAEC-treated ventilated lambs (n = 6). # p

    Techniques Used: Expressing

    18) Product Images from "An extracatalytic function of CD45 in B cells is mediated by CD22"

    Article Title: An extracatalytic function of CD45 in B cells is mediated by CD22

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

    doi: 10.1073/pnas.1519925112

    CS and ΔC transgenes regulate B-cell development independently of endogenous CD45. ( A ) splenocytes from CD45 −/− mice with or without CS or ΔC Tg expression were stained to detect CD21, CD23, IgM, and IgD. Upper depicts
    Figure Legend Snippet: CS and ΔC transgenes regulate B-cell development independently of endogenous CD45. ( A ) splenocytes from CD45 −/− mice with or without CS or ΔC Tg expression were stained to detect CD21, CD23, IgM, and IgD. Upper depicts

    Techniques Used: Mouse Assay, Expressing, Staining

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    Cytometry:

    Article Title: IL35 hinders endogenous antitumor T-cell immunity and responsiveness to immunotherapy in pancreatic cancer
    Article Snippet: .. The following monoclonal antibodies directed against mouse antigens were used for flow cytometry: Biolegend - CD45 (clone 30-F11), CD11b (clone M1/70), F4/80 (clone BM8), Ly6G (clone 1A8), Ly6C (clone HK1.4), CD206 (clone C068C2), MHCII (clone M5/114.15.2), CD3 (clone 17A2), CD4 (clone GK1.5), IFNγ (clone XMG1.2), TNFα (clone MP6-XT22), Foxp3 (clone FJK-16S), CD8 (clone 53–6.7), IL12p40 (clone C15.6), IL27p28 (clone MM27–7B1), CD19 (clone 6D5), CD45RB (clone C363–16A), CD21 (clone 7E9); Ebioscience - IL35p35 (clone 4D10p35); R & D - IL35EBi3 (clone 355022); BD Biosciences - CD1d (clone 1B1). .. Viability of cells were determined by staining with either 7-aminoactinomycin (Biolegend) or Live/Dead Aqua cell stain kit (Life technologies).

    Double Staining:

    Article Title: Molecular cloning of porcine Siglec-3, Siglec-5 and Siglec-10, and identification of Siglec-10 as an alternative receptor for porcine reproductive and respiratory syndrome virus (PRRSV)
    Article Snippet: .. For double staining of B cells and Siglec-10 positive cells, primary mouse monoclonal antibodies against the B cell marker CD21 (IgG2b) and Siglec-10 (1G10, IgG1) and isotype-specific secondary antibodies conjugated with FITC or Alexa Fluor 594 (Invitrogen) were used. .. Cell nuclei were stained with Hoechst 33 342.

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  • 94
    Thermo Fisher anti cd21 35
    Anti-CD137 mAb Results in Disorganization of B Cell Follicle Architecture in the Spleen Four-week-old WT C57BL/6 male mice were inoculated with 10 3 FFU of CHIKV. At 2 dpi, 400 μg of agonistic anti-CD137 or isotype control mAb was administered by an i.p. route. Spleens were harvested at 6 dpi (A–D), 7 dpi (E and F), and 14 dpi (I–M) for imaging. (A–D) FDCs (green) were stained for <t>CD21/35;</t> IgD + B cells (red); and T cell zone (turquoise), CCL21. (B and D) Insets of the respective dotted boxes. (E and F) FDCs (green) were stained for CD21/35; IgD + B cells (red); and CD4 + T cells (snow). White scale bars indicate 50 μm. Yellow arrows indicate IgD + B cells surrounding FDCs, and white arrows indicate IgD + B cells at the CCL21 + T cell zone border. (G and H) The number of CD45 − CD21/35 + CD54 + FDCs in the spleen at 7 and 14 dpi was analyzed by flow cytometry. (G) Representative flow cytometry dot plots of FDCs are shown. (I) B cells (blue) were stained for B220; FDCs (green), CD21/35; and GC B cells (snow), GL7. White scale bars indicate 500 μm or 100 μm (insets). Each symbol represents an individual FDC-containing follicle (J), GC B cell (K), or spleen (L), and bars indicate mean values. Quantification was performed for FDC area per FDC-containing-follicle (J), distance between the closest GC B cells (K), and GC B cells greater than 10 μm from an FDC (L). (M) Left and middle: FDCs (blue) were stained for CD21/35; VCAM-1 (red); merge (white). (M) Right: GC B cells (white) were stained for GL7; VCAM-1 + FDCs (red). White scale bars indicate 200 μm or 50 μm (insets). The images are representative of 3 spleens per group from 2 independent experiments (Mann-Whitney test: ***p
    Anti Cd21 35, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher cd21 immunostaining
    Morphological and immunohistochemical analyses of the follicular lymphoma. a Infiltration of the lymph node by a nodular proliferation. × 10 lens objective. b Nodules composed of centrocytes. × 40 lens objective. c Bcl-2 expression by the tumor cells. × 10 lens objective. d <t>CD21</t> <t>immunostaining</t> highlighting the follicular dendritic cell meshwork. × 10 lens objective
    Cd21 Immunostaining, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd21 immunostaining/product/Thermo Fisher
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    99
    Thermo Fisher mouse anti human cd21
    B cell accumulation and activation in the lungs after BeO exposure in FVB/N HLA-DP2–Tg mice. HLA-DP2–Tg FVB/N mice were exposed to PBS or BeO as described in Methods, and BAL was harvested on day 21 and analyzed by flow cytometry. ( A ) Representative density plots show the percentage of CD19 + B cells and CD3 + T cells in the BAL of PBS- (left) and BeO-exposed (right) mice. The numbers in each density plot indicate the percentage of cells in each population. ( B ) Percentage of lymphocytes that are CD3 + T cells and CD19 + B cells in the BAL are cumulatively shown for PBS- and BeO-exposed mice. ( C ) Number of CD19 + B cells in the BAL of HLA-DP2–Tg mice exposed to either PBS or BeO is shown. ( D ) B220 + CD19 + B cells in spleen were stained for <t>CD21</t> and CD23 expression to establish gates for marginal zone B cells (CD21 + CD23 – ) and follicular B cells (CD23 + CD21 – ) in BAL and lung-draining lymph nodes (LDLNs). The numbers in each gate indicate the percentage of cells in each B cell subset. ( E ) Percentage of CD23 + and CD23 – B cells in the spleen and BAL of HLA-DP2–Tg mice after BeO exposure is shown. The expression (mean fluorescence intensity [MFI]) of costimulatory molecules CD86 ( F ) and CD40 ( G ), and HLA-DP ( H ) on CD19 + CD23 + and CD19 + CD23 – B cells derived from spleen ( n = 10 mice/group) and BAL ( n = 6 mice/group) of HLA-DP2–Tg mice after BeO exposure is shown. Data are representative of 3 independent experiments having 5 to 10 mice per group. Solid lines and error bars depict the mean ± SEM. Student’s t test (2 tailed) ( B and C ) and 2-way ANOVA were used to test for differences. A P value of
    Mouse Anti Human Cd21, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Anti-CD137 mAb Results in Disorganization of B Cell Follicle Architecture in the Spleen Four-week-old WT C57BL/6 male mice were inoculated with 10 3 FFU of CHIKV. At 2 dpi, 400 μg of agonistic anti-CD137 or isotype control mAb was administered by an i.p. route. Spleens were harvested at 6 dpi (A–D), 7 dpi (E and F), and 14 dpi (I–M) for imaging. (A–D) FDCs (green) were stained for CD21/35; IgD + B cells (red); and T cell zone (turquoise), CCL21. (B and D) Insets of the respective dotted boxes. (E and F) FDCs (green) were stained for CD21/35; IgD + B cells (red); and CD4 + T cells (snow). White scale bars indicate 50 μm. Yellow arrows indicate IgD + B cells surrounding FDCs, and white arrows indicate IgD + B cells at the CCL21 + T cell zone border. (G and H) The number of CD45 − CD21/35 + CD54 + FDCs in the spleen at 7 and 14 dpi was analyzed by flow cytometry. (G) Representative flow cytometry dot plots of FDCs are shown. (I) B cells (blue) were stained for B220; FDCs (green), CD21/35; and GC B cells (snow), GL7. White scale bars indicate 500 μm or 100 μm (insets). Each symbol represents an individual FDC-containing follicle (J), GC B cell (K), or spleen (L), and bars indicate mean values. Quantification was performed for FDC area per FDC-containing-follicle (J), distance between the closest GC B cells (K), and GC B cells greater than 10 μm from an FDC (L). (M) Left and middle: FDCs (blue) were stained for CD21/35; VCAM-1 (red); merge (white). (M) Right: GC B cells (white) were stained for GL7; VCAM-1 + FDCs (red). White scale bars indicate 200 μm or 50 μm (insets). The images are representative of 3 spleens per group from 2 independent experiments (Mann-Whitney test: ***p

    Journal: Cell reports. Medicine

    Article Title: An Agonistic Anti-CD137 Antibody Disrupts Lymphoid Follicle Structure and T-Cell-Dependent Antibody Responses

    doi: 10.1016/j.xcrm.2020.100035

    Figure Lengend Snippet: Anti-CD137 mAb Results in Disorganization of B Cell Follicle Architecture in the Spleen Four-week-old WT C57BL/6 male mice were inoculated with 10 3 FFU of CHIKV. At 2 dpi, 400 μg of agonistic anti-CD137 or isotype control mAb was administered by an i.p. route. Spleens were harvested at 6 dpi (A–D), 7 dpi (E and F), and 14 dpi (I–M) for imaging. (A–D) FDCs (green) were stained for CD21/35; IgD + B cells (red); and T cell zone (turquoise), CCL21. (B and D) Insets of the respective dotted boxes. (E and F) FDCs (green) were stained for CD21/35; IgD + B cells (red); and CD4 + T cells (snow). White scale bars indicate 50 μm. Yellow arrows indicate IgD + B cells surrounding FDCs, and white arrows indicate IgD + B cells at the CCL21 + T cell zone border. (G and H) The number of CD45 − CD21/35 + CD54 + FDCs in the spleen at 7 and 14 dpi was analyzed by flow cytometry. (G) Representative flow cytometry dot plots of FDCs are shown. (I) B cells (blue) were stained for B220; FDCs (green), CD21/35; and GC B cells (snow), GL7. White scale bars indicate 500 μm or 100 μm (insets). Each symbol represents an individual FDC-containing follicle (J), GC B cell (K), or spleen (L), and bars indicate mean values. Quantification was performed for FDC area per FDC-containing-follicle (J), distance between the closest GC B cells (K), and GC B cells greater than 10 μm from an FDC (L). (M) Left and middle: FDCs (blue) were stained for CD21/35; VCAM-1 (red); merge (white). (M) Right: GC B cells (white) were stained for GL7; VCAM-1 + FDCs (red). White scale bars indicate 200 μm or 50 μm (insets). The images are representative of 3 spleens per group from 2 independent experiments (Mann-Whitney test: ***p

    Article Snippet: 18-μm sections were cut on a Lecia cryostat (Leica Microsystems), blocked with 10% bovine and donkey serum, and then stained with combinations of the following antibodies: anti-reticular fibroblasts and reticular fibers (ab51824, Abcam), anti-CD21/35 (48–0212-82, Thermo), anti-CD4 (ab183685, Abcam), Alexa555-conjugated anti-rabbit IgG (A-21428, Thermo), Alexa700-conjugated anti-B220 (103232, Biolegend), Alexa647-conjugated anti-GL7 (144606, Biolegend), Alexa647-conjugated anti-goat IgG (A-21447, Thermo), biotinylated anti-IgD (1120–08, SouthernBiotech) antibody and Alexa555-conjugated streptavidin (S32355, Thermo).

    Techniques: Mouse Assay, Imaging, Staining, Flow Cytometry, MANN-WHITNEY

    Morphological and immunohistochemical analyses of the follicular lymphoma. a Infiltration of the lymph node by a nodular proliferation. × 10 lens objective. b Nodules composed of centrocytes. × 40 lens objective. c Bcl-2 expression by the tumor cells. × 10 lens objective. d CD21 immunostaining highlighting the follicular dendritic cell meshwork. × 10 lens objective

    Journal: Journal of Medical Case Reports

    Article Title: Transformation of a low-grade follicular lymphoma into a composite lymphoma combining a high-grade B-cell lymphoma and a lymphoblastic neoplasm expressing Terminal deoxynucleotidyl Transferase: a case report

    doi: 10.1186/s13256-020-02433-6

    Figure Lengend Snippet: Morphological and immunohistochemical analyses of the follicular lymphoma. a Infiltration of the lymph node by a nodular proliferation. × 10 lens objective. b Nodules composed of centrocytes. × 40 lens objective. c Bcl-2 expression by the tumor cells. × 10 lens objective. d CD21 immunostaining highlighting the follicular dendritic cell meshwork. × 10 lens objective

    Article Snippet: The CD21 immunostaining highlighted the follicular dendritic cell meshwork (Fig. d).

    Techniques: Immunohistochemistry, Expressing, Immunostaining

    B cell accumulation and activation in the lungs after BeO exposure in FVB/N HLA-DP2–Tg mice. HLA-DP2–Tg FVB/N mice were exposed to PBS or BeO as described in Methods, and BAL was harvested on day 21 and analyzed by flow cytometry. ( A ) Representative density plots show the percentage of CD19 + B cells and CD3 + T cells in the BAL of PBS- (left) and BeO-exposed (right) mice. The numbers in each density plot indicate the percentage of cells in each population. ( B ) Percentage of lymphocytes that are CD3 + T cells and CD19 + B cells in the BAL are cumulatively shown for PBS- and BeO-exposed mice. ( C ) Number of CD19 + B cells in the BAL of HLA-DP2–Tg mice exposed to either PBS or BeO is shown. ( D ) B220 + CD19 + B cells in spleen were stained for CD21 and CD23 expression to establish gates for marginal zone B cells (CD21 + CD23 – ) and follicular B cells (CD23 + CD21 – ) in BAL and lung-draining lymph nodes (LDLNs). The numbers in each gate indicate the percentage of cells in each B cell subset. ( E ) Percentage of CD23 + and CD23 – B cells in the spleen and BAL of HLA-DP2–Tg mice after BeO exposure is shown. The expression (mean fluorescence intensity [MFI]) of costimulatory molecules CD86 ( F ) and CD40 ( G ), and HLA-DP ( H ) on CD19 + CD23 + and CD19 + CD23 – B cells derived from spleen ( n = 10 mice/group) and BAL ( n = 6 mice/group) of HLA-DP2–Tg mice after BeO exposure is shown. Data are representative of 3 independent experiments having 5 to 10 mice per group. Solid lines and error bars depict the mean ± SEM. Student’s t test (2 tailed) ( B and C ) and 2-way ANOVA were used to test for differences. A P value of

    Journal: JCI Insight

    Article Title: Protective role of B cells in sterile particulate–induced lung injury

    doi: 10.1172/jci.insight.125494

    Figure Lengend Snippet: B cell accumulation and activation in the lungs after BeO exposure in FVB/N HLA-DP2–Tg mice. HLA-DP2–Tg FVB/N mice were exposed to PBS or BeO as described in Methods, and BAL was harvested on day 21 and analyzed by flow cytometry. ( A ) Representative density plots show the percentage of CD19 + B cells and CD3 + T cells in the BAL of PBS- (left) and BeO-exposed (right) mice. The numbers in each density plot indicate the percentage of cells in each population. ( B ) Percentage of lymphocytes that are CD3 + T cells and CD19 + B cells in the BAL are cumulatively shown for PBS- and BeO-exposed mice. ( C ) Number of CD19 + B cells in the BAL of HLA-DP2–Tg mice exposed to either PBS or BeO is shown. ( D ) B220 + CD19 + B cells in spleen were stained for CD21 and CD23 expression to establish gates for marginal zone B cells (CD21 + CD23 – ) and follicular B cells (CD23 + CD21 – ) in BAL and lung-draining lymph nodes (LDLNs). The numbers in each gate indicate the percentage of cells in each B cell subset. ( E ) Percentage of CD23 + and CD23 – B cells in the spleen and BAL of HLA-DP2–Tg mice after BeO exposure is shown. The expression (mean fluorescence intensity [MFI]) of costimulatory molecules CD86 ( F ) and CD40 ( G ), and HLA-DP ( H ) on CD19 + CD23 + and CD19 + CD23 – B cells derived from spleen ( n = 10 mice/group) and BAL ( n = 6 mice/group) of HLA-DP2–Tg mice after BeO exposure is shown. Data are representative of 3 independent experiments having 5 to 10 mice per group. Solid lines and error bars depict the mean ± SEM. Student’s t test (2 tailed) ( B and C ) and 2-way ANOVA were used to test for differences. A P value of

    Article Snippet: FDCs in human lungs were detected with mouse anti–human CD21 (ThermoFisher Scientific, 2G9).

    Techniques: Activation Assay, Mouse Assay, Flow Cytometry, Cytometry, Staining, Expressing, Fluorescence, Derivative Assay