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SouthernBiotech anti igm
Flow cytometric analysis of lymphocyte populations in BCAP −/− mice. (A) Expression of BCAP in splenic B cells. Spleen cells stained for CD3 and B220 were analyzed by flow cytometric intracellular staining with an antiserum to BCAP plus goat anti–rabbit IgG-FITC (thick solid line, wild-type mice; shaded area, BCAP −/− mice). Data shown are representative of three independent experiments. (B) Single-cell suspensions from thymus (Thy), bone marrow (BM), lymph node (LN), spleen (SP), and peritoneal cavity (PerC), were stained with the indicated Abs and analyzed using a FACScan ® (wt, wild-type mice). Numbers indicate the percentages of lymphoid cells in the quadrants or enclosed areas. <t>IgM</t> versus IgD and CD21 versus <t>CD23</t> profiles are shown for B220 + cells, and CD11b versus CD16 profile for B220 − cells. Data shown are representative of six independent experiments.
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1) Product Images from "Essential Immunoregulatory Role for BCAP in B Cell Development and Function"

Article Title: Essential Immunoregulatory Role for BCAP in B Cell Development and Function

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20011751

Flow cytometric analysis of lymphocyte populations in BCAP −/− mice. (A) Expression of BCAP in splenic B cells. Spleen cells stained for CD3 and B220 were analyzed by flow cytometric intracellular staining with an antiserum to BCAP plus goat anti–rabbit IgG-FITC (thick solid line, wild-type mice; shaded area, BCAP −/− mice). Data shown are representative of three independent experiments. (B) Single-cell suspensions from thymus (Thy), bone marrow (BM), lymph node (LN), spleen (SP), and peritoneal cavity (PerC), were stained with the indicated Abs and analyzed using a FACScan ® (wt, wild-type mice). Numbers indicate the percentages of lymphoid cells in the quadrants or enclosed areas. IgM versus IgD and CD21 versus CD23 profiles are shown for B220 + cells, and CD11b versus CD16 profile for B220 − cells. Data shown are representative of six independent experiments.
Figure Legend Snippet: Flow cytometric analysis of lymphocyte populations in BCAP −/− mice. (A) Expression of BCAP in splenic B cells. Spleen cells stained for CD3 and B220 were analyzed by flow cytometric intracellular staining with an antiserum to BCAP plus goat anti–rabbit IgG-FITC (thick solid line, wild-type mice; shaded area, BCAP −/− mice). Data shown are representative of three independent experiments. (B) Single-cell suspensions from thymus (Thy), bone marrow (BM), lymph node (LN), spleen (SP), and peritoneal cavity (PerC), were stained with the indicated Abs and analyzed using a FACScan ® (wt, wild-type mice). Numbers indicate the percentages of lymphoid cells in the quadrants or enclosed areas. IgM versus IgD and CD21 versus CD23 profiles are shown for B220 + cells, and CD11b versus CD16 profile for B220 − cells. Data shown are representative of six independent experiments.

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

Impaired proliferative response of BCAP −/− splenic B cells. (A) Purified splenic B cells from wild-type and BCAP −/− mice were cultured with medium, F(ab′) 2 goat anti-IgM Ab (15 μg/ml), rat anti-CD40 Ab (10 μg/ml), or LPS (10 μg/ml). The mean and standard deviations are plotted for wild-type (black bars) and BCAP −/− splenic B cells (white bars). Experiments were performed in triplicates. Data shown are representative of three independent experiments. (B) Splenic B cells from the indicated mice were sorted into B220 + HSA hi (immature B) and B220 + HSA lo (mature B) subsets, and experiments were performed in triplicates. Data shown are representative of three independent experiments.
Figure Legend Snippet: Impaired proliferative response of BCAP −/− splenic B cells. (A) Purified splenic B cells from wild-type and BCAP −/− mice were cultured with medium, F(ab′) 2 goat anti-IgM Ab (15 μg/ml), rat anti-CD40 Ab (10 μg/ml), or LPS (10 μg/ml). The mean and standard deviations are plotted for wild-type (black bars) and BCAP −/− splenic B cells (white bars). Experiments were performed in triplicates. Data shown are representative of three independent experiments. (B) Splenic B cells from the indicated mice were sorted into B220 + HSA hi (immature B) and B220 + HSA lo (mature B) subsets, and experiments were performed in triplicates. Data shown are representative of three independent experiments.

Techniques Used: Purification, Mouse Assay, Cell Culture

2) Product Images from "Mutations in the RAS-BRAF-MAPK-ERK pathway define a specific subgroup of patients with adverse clinical features and provide new therapeutic options in chronic lymphocytic leukemia"

Article Title: Mutations in the RAS-BRAF-MAPK-ERK pathway define a specific subgroup of patients with adverse clinical features and provide new therapeutic options in chronic lymphocytic leukemia

Journal: Haematologica

doi: 10.3324/haematol.2018.196931

Effect of RAS-BRAF-MAPK-ERK inhibitors in cases of RAS-BRAF-MAPK-ERK-mutated and unmutated IGHV chronic lymphocytic leukemia. (A) Cells from 17 cases of chronic lymphocytic leukemia (CLL), nine containing mutations in the RAS-BRAF-MAPK-ERK pathway ( KITLG , PTPN11 , KRAS , BRAF , MAPK1 , MAP2K1 , MAP2K2 ) and eight with unmutated IGHV genes (U-IGHV) with no alterations in genes of the RAS-BRAF-MAPK-ERK pathway were treated with vemurafenib 2.5 μM or dabrafenib 2.5 μM. p-ERK levels were analyzed by flow cytometry after 1.5 h of treatment and expressed relative to untreated cells (Ct) at basal levels (unstimulated) or after stimulation with anti-IgM (stimulated) (* P
Figure Legend Snippet: Effect of RAS-BRAF-MAPK-ERK inhibitors in cases of RAS-BRAF-MAPK-ERK-mutated and unmutated IGHV chronic lymphocytic leukemia. (A) Cells from 17 cases of chronic lymphocytic leukemia (CLL), nine containing mutations in the RAS-BRAF-MAPK-ERK pathway ( KITLG , PTPN11 , KRAS , BRAF , MAPK1 , MAP2K1 , MAP2K2 ) and eight with unmutated IGHV genes (U-IGHV) with no alterations in genes of the RAS-BRAF-MAPK-ERK pathway were treated with vemurafenib 2.5 μM or dabrafenib 2.5 μM. p-ERK levels were analyzed by flow cytometry after 1.5 h of treatment and expressed relative to untreated cells (Ct) at basal levels (unstimulated) or after stimulation with anti-IgM (stimulated) (* P

Techniques Used: Flow Cytometry, Cytometry

3) Product Images from "Maternal T cells limit engraftment after in utero hematopoietic cell transplantation in mice"

Article Title: Maternal T cells limit engraftment after in utero hematopoietic cell transplantation in mice

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI44907

The rejection of in utero transplanted allogeneic hematopoietic cells occurs independent of maternal B cells and maternal alloantibodies. ( A ) Frequency of chimerism after IUHCTx of B6 FL cells into fetuses born to a wild-type BALB/c father and either a wild-type BALB/c mother ( n = 43) or a B cell–deficient (JHD) mother ( n = 9). ( B ) Change in levels of chimerism over time in engrafted animals when normalized to the initial level of chimerism at 4 weeks after transplantation (BALB/c mother, n = 10; JHD mother, n = 4). ( C ) Frequency of chimerism in pups fostered by naive mothers (BALB/c fostered, n = 20) and in non-fostered pups (BALB/c, n = 43) after IUHCTx with B6 FL. ( D ) Serum from BALB/c mothers whose fetuses received allogeneic IUHCTx ( n ≥ 10) was analyzed by flow cytometry to quantify total serum IgM (left panels) and IgG (right panels) alloantibody. Comparison groups include naive ( n = 7) and sensitized (Sens., n ≥ 3) mice. ( E ) Total IgM (left panel) and IgG (right panel) alloantibody production at 1, 2, 4, and 6 weeks after sensitization is shown as the MFI relative to a no-serum sample (relative MFI). * P
Figure Legend Snippet: The rejection of in utero transplanted allogeneic hematopoietic cells occurs independent of maternal B cells and maternal alloantibodies. ( A ) Frequency of chimerism after IUHCTx of B6 FL cells into fetuses born to a wild-type BALB/c father and either a wild-type BALB/c mother ( n = 43) or a B cell–deficient (JHD) mother ( n = 9). ( B ) Change in levels of chimerism over time in engrafted animals when normalized to the initial level of chimerism at 4 weeks after transplantation (BALB/c mother, n = 10; JHD mother, n = 4). ( C ) Frequency of chimerism in pups fostered by naive mothers (BALB/c fostered, n = 20) and in non-fostered pups (BALB/c, n = 43) after IUHCTx with B6 FL. ( D ) Serum from BALB/c mothers whose fetuses received allogeneic IUHCTx ( n ≥ 10) was analyzed by flow cytometry to quantify total serum IgM (left panels) and IgG (right panels) alloantibody. Comparison groups include naive ( n = 7) and sensitized (Sens., n ≥ 3) mice. ( E ) Total IgM (left panel) and IgG (right panel) alloantibody production at 1, 2, 4, and 6 weeks after sensitization is shown as the MFI relative to a no-serum sample (relative MFI). * P

Techniques Used: In Utero, Transplantation Assay, Flow Cytometry, Cytometry, Mouse Assay

4) Product Images from "The Absence of Tssc6, a Member of the Tetraspanin Superfamily, Does Not Affect Lymphoid Development but Enhances In Vitro T-Cell Proliferative Responses"

Article Title: The Absence of Tssc6, a Member of the Tetraspanin Superfamily, Does Not Affect Lymphoid Development but Enhances In Vitro T-Cell Proliferative Responses

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.14.5006-5018.2002

Humoral response of Tssc6 gt/gt mice to immunization with T-cell-dependent and T-cell-independent antigens. The T-cell-dependent antigen NP 18 -KLH was administered i.p. (arrowheads) at the following doses for the primary response. (A) One hundred micrograms of NP 18 -KLH precipitated in alum. (B) Ten micrograms of NP 18 -KLH precipitated in alum. (C) One microgram of NP 18 -KLH precipitated in alum. (D) Twenty micrograms of NP 18 -KLH without adjuvant. Boosting occurred where indicated by the second arrow. (E) Fifty micrograms of NP-LPS injected i.p. on day 0 into each mouse to generate a T-cell-independent immune response. Anti-NP IgM and IgG3 were measured by ELISA at the indicated time points. Data represent the means and standard errors of the means for six mice (panels A through D) or cohorts of three mice (panel E) of each genotype.
Figure Legend Snippet: Humoral response of Tssc6 gt/gt mice to immunization with T-cell-dependent and T-cell-independent antigens. The T-cell-dependent antigen NP 18 -KLH was administered i.p. (arrowheads) at the following doses for the primary response. (A) One hundred micrograms of NP 18 -KLH precipitated in alum. (B) Ten micrograms of NP 18 -KLH precipitated in alum. (C) One microgram of NP 18 -KLH precipitated in alum. (D) Twenty micrograms of NP 18 -KLH without adjuvant. Boosting occurred where indicated by the second arrow. (E) Fifty micrograms of NP-LPS injected i.p. on day 0 into each mouse to generate a T-cell-independent immune response. Anti-NP IgM and IgG3 were measured by ELISA at the indicated time points. Data represent the means and standard errors of the means for six mice (panels A through D) or cohorts of three mice (panel E) of each genotype.

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

5) Product Images from "Hematopoietic Overexpression of FOG1 Does Not Affect B-Cells but Reduces the Number of Circulating Eosinophils"

Article Title: Hematopoietic Overexpression of FOG1 Does Not Affect B-Cells but Reduces the Number of Circulating Eosinophils

Journal: PLoS ONE

doi: 10.1371/journal.pone.0092836

Overexpression of FOG-1 in mature B-cells. A . In R26 FOG-1 :Cd23-Cre mice, hCD2t expression is restricted to mature B-cells. Cell surface expression of hCD2t was analyzed by flow cytometry in Pre-B-cells (B220+, CD25+) and in mature B-cells (B220+, IgM high ) of control (R26 FOG-1 ) and R26 FOG-1 :Cd23-Cre mice. Representative results of at least 3 independent experiments are shown. B . FOG-1 mRNA is increased 3-fold in R26 FOG-1 :Cd23-Cre mature B-cells. RNA extracted from 3 control (R26 FOG-1 ) and 3 R26 FOG-1 :Cd23-Cre mice was reverse transcribed and subjected to quantitative PCR to detect FOG-1 and RNA Polymerase II (RPII, for normalization) transcripts. Standard error of the mean is shown. C . FOG-1 protein is up-regulated ca. 6-fold in mature B-cells derived from R26 FOG-1 :Cd23-Cre mice. FOG-1 and actin proteins were detected by western blotting in mature B-cells derived from 3 control (R26 FOG-1 ) and 3 R26 FOG-1 :Cd23-Cre mice (upper panel). The band intensities were quantified by LiCor Odyssey scanning and normalized to expression of actin (lower panel). Standard error of the mean is shown. D . FOG-1-overexpressing mature B-cells respond normally to in vitro stimulation. Splenic resting mature B-cells isolated from 3 to 6 control (R26 FOG-1 , black dots) or R26 FOG-1 :Cd23-Cre mice (grey dots) were activated in vitro by LPS, LPS+IL4 or anti-CD40+IL4 for 4 days. Titers of IgM (left panel) or IgG1 (right panel) in the culture supernatants were determined by ELISA; means are shown (red bar) as well as the corresponding p values at the bottom.
Figure Legend Snippet: Overexpression of FOG-1 in mature B-cells. A . In R26 FOG-1 :Cd23-Cre mice, hCD2t expression is restricted to mature B-cells. Cell surface expression of hCD2t was analyzed by flow cytometry in Pre-B-cells (B220+, CD25+) and in mature B-cells (B220+, IgM high ) of control (R26 FOG-1 ) and R26 FOG-1 :Cd23-Cre mice. Representative results of at least 3 independent experiments are shown. B . FOG-1 mRNA is increased 3-fold in R26 FOG-1 :Cd23-Cre mature B-cells. RNA extracted from 3 control (R26 FOG-1 ) and 3 R26 FOG-1 :Cd23-Cre mice was reverse transcribed and subjected to quantitative PCR to detect FOG-1 and RNA Polymerase II (RPII, for normalization) transcripts. Standard error of the mean is shown. C . FOG-1 protein is up-regulated ca. 6-fold in mature B-cells derived from R26 FOG-1 :Cd23-Cre mice. FOG-1 and actin proteins were detected by western blotting in mature B-cells derived from 3 control (R26 FOG-1 ) and 3 R26 FOG-1 :Cd23-Cre mice (upper panel). The band intensities were quantified by LiCor Odyssey scanning and normalized to expression of actin (lower panel). Standard error of the mean is shown. D . FOG-1-overexpressing mature B-cells respond normally to in vitro stimulation. Splenic resting mature B-cells isolated from 3 to 6 control (R26 FOG-1 , black dots) or R26 FOG-1 :Cd23-Cre mice (grey dots) were activated in vitro by LPS, LPS+IL4 or anti-CD40+IL4 for 4 days. Titers of IgM (left panel) or IgG1 (right panel) in the culture supernatants were determined by ELISA; means are shown (red bar) as well as the corresponding p values at the bottom.

Techniques Used: Over Expression, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Derivative Assay, Western Blot, In Vitro, Isolation, Enzyme-linked Immunosorbent Assay

Normal B-cell, T-cell and granular cell populations in R26FOG-1:Vav-iCre mice. A . Cells of the bone marrow (BM), spleen (Spl) and thymus (Thy) of R26 FOG-1 (black bars) and R26 FOG-1 :Vav-iCre (grey bars) mice were enumerated. Standard error of the mean is shown. B . Bone marrow cells were stained with anti-B220 and anti-IgM antibodies to analyze B-cell development. C . Splenocytes were stained with anti-B220 and anti-IgM antibodies to identify B-cells. D . Thymocytes were stained with anti-CD4 and anti-CD8 antibodies to analyze T-cell development. E . Splenocytes were stained with anti-CD4 and anti-CD8 antibodies to identify mature T-cells. F . Bone marrow cells were stained with anti-TER119 and anti-CD71 antibodies to analyze erythropoiesis. G . Bone marrow cells were stained with anti-Gr1 and anti-CD11b antibodies to identify Gr1+ CD11b+ myeloid cells. H . Splenocytes were stained with anti-TER119 and anti-CD71 antibodies to analyze splenic erythropoiesis. Cells were analyzed by flow cytometry in R26 FOG-1 (control) and R26 FOG-1 :Vav-iCre animals; data for one representative animal are shown (n = 5 for each genotype). Percentages of the populations are shown next to the gates. A diagram representing the developmental pathway of the different lineages from pale (progenitors) to dark grey (differentiated cells) is shown next to the pseudo-dotplots B, D, F and H.
Figure Legend Snippet: Normal B-cell, T-cell and granular cell populations in R26FOG-1:Vav-iCre mice. A . Cells of the bone marrow (BM), spleen (Spl) and thymus (Thy) of R26 FOG-1 (black bars) and R26 FOG-1 :Vav-iCre (grey bars) mice were enumerated. Standard error of the mean is shown. B . Bone marrow cells were stained with anti-B220 and anti-IgM antibodies to analyze B-cell development. C . Splenocytes were stained with anti-B220 and anti-IgM antibodies to identify B-cells. D . Thymocytes were stained with anti-CD4 and anti-CD8 antibodies to analyze T-cell development. E . Splenocytes were stained with anti-CD4 and anti-CD8 antibodies to identify mature T-cells. F . Bone marrow cells were stained with anti-TER119 and anti-CD71 antibodies to analyze erythropoiesis. G . Bone marrow cells were stained with anti-Gr1 and anti-CD11b antibodies to identify Gr1+ CD11b+ myeloid cells. H . Splenocytes were stained with anti-TER119 and anti-CD71 antibodies to analyze splenic erythropoiesis. Cells were analyzed by flow cytometry in R26 FOG-1 (control) and R26 FOG-1 :Vav-iCre animals; data for one representative animal are shown (n = 5 for each genotype). Percentages of the populations are shown next to the gates. A diagram representing the developmental pathway of the different lineages from pale (progenitors) to dark grey (differentiated cells) is shown next to the pseudo-dotplots B, D, F and H.

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

6) Product Images from "Neonatal Exposure to Pneumococcal Phosphorylcholine Modulates the Development of House Dust Mite Allergy during Adult Life"

Article Title: Neonatal Exposure to Pneumococcal Phosphorylcholine Modulates the Development of House Dust Mite Allergy during Adult Life

Journal: The Journal of Immunology Author Choice

doi: 10.4049/jimmunol.1500251

Anti-PC Abs bind both HDM and R36A, and neonatal exposure to PC-bearing R36A reduced cellular infiltration into the bronchoalveolar space following adult exposure to HDM. Anti-PC IgM (BH8), anti-PC IgA (S107) Abs, or their respective isotype control Abs were incubated with ( A ) pneumococcal bacterial strains PC-bearing R36A or ( B ) PC-deficient JY2190 or ( C ) with processed HDM allergen. (A–C) Ab binding was detected by flow cytometry. Whole mounts of HDM were stained with ( D ) isotype control Ab or ( E ) fluorescently labeled anti-PC IgM Ab (BH8) and viewed with a Leica/Leitz DMRB microscope. ( F ) Littermate C57BL/6 mice that were either treated i.p. with PBS, or immunized with JY2190 or R36A at day 3 of life, as well as the T15 KI mice were sensitized with HDM on day 0 and challenged with HDM daily on days 7–11 at 6–8 wk of age. Following challenge with HDM, mice were euthanized on day 14, tracheas were cannulated, and a 5-ml wash of the bronchoalveolar space was removed. ( G and H . ( I – L ) Equal volumes of BALF were cytocentrifuged onto glass slides and stained with modified Wright stain to identify alveolar macrophages (A.M.), eosinophils (eo), and neutrophils (neut). Values represent the mean ± SEM from five independent experiments with 5–10 mice per group. Data were analyzed by ANOVA, in which statistically significant results are represented as ** p
Figure Legend Snippet: Anti-PC Abs bind both HDM and R36A, and neonatal exposure to PC-bearing R36A reduced cellular infiltration into the bronchoalveolar space following adult exposure to HDM. Anti-PC IgM (BH8), anti-PC IgA (S107) Abs, or their respective isotype control Abs were incubated with ( A ) pneumococcal bacterial strains PC-bearing R36A or ( B ) PC-deficient JY2190 or ( C ) with processed HDM allergen. (A–C) Ab binding was detected by flow cytometry. Whole mounts of HDM were stained with ( D ) isotype control Ab or ( E ) fluorescently labeled anti-PC IgM Ab (BH8) and viewed with a Leica/Leitz DMRB microscope. ( F ) Littermate C57BL/6 mice that were either treated i.p. with PBS, or immunized with JY2190 or R36A at day 3 of life, as well as the T15 KI mice were sensitized with HDM on day 0 and challenged with HDM daily on days 7–11 at 6–8 wk of age. Following challenge with HDM, mice were euthanized on day 14, tracheas were cannulated, and a 5-ml wash of the bronchoalveolar space was removed. ( G and H . ( I – L ) Equal volumes of BALF were cytocentrifuged onto glass slides and stained with modified Wright stain to identify alveolar macrophages (A.M.), eosinophils (eo), and neutrophils (neut). Values represent the mean ± SEM from five independent experiments with 5–10 mice per group. Data were analyzed by ANOVA, in which statistically significant results are represented as ** p

Techniques Used: Incubation, Binding Assay, Flow Cytometry, Cytometry, Staining, Labeling, Microscopy, Mouse Assay, Modification, Wright Stain

7) Product Images from "Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells"

Article Title: Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells

Journal: The EMBO Journal

doi: 10.15252/embj.201797980

Fitness of Ramos cells depends on the Igβ subunit of the BCR A schematic diagram showing the route for the generation of single‐ and multi‐BCR components KO from wild‐type (WT) Ramos B cells by the CRISPR/Cas9 method. Cell proliferation assay of WT and Ramos‐null cells using CytoTell™ UltraGreen. Null = HC/LC/Igα/Igβ tetra KO. A diagram depicting the growth competition assay. WT Ramos cells were retrovirally transduced with pMIG empty vector (EV) to create GFP‐labeled WT cells (gWT). BCR component KO cells were mixed together with gWT Ramos cells at about 1:1 ratio at day 0, and the relative amount of GFP − BCR components KO cells was then measured by flow cytometry at different time points. The expression levels of IgM‐BCR and IgD‐BCR on the surface of WT and Ramos‐null cells were determined by flow cytometry. Growth competition between Ramos‐null and gWT cells. The competition between GFP − WT and gWT cells serves as a control. The data represent the mean and standard error of a minimum of three independent experiments. The expression levels of IgM‐BCR and IgD‐BCR on the surface of WT and BCR component KO Ramos cells were determined by flow cytometry. Growth competition of the BCR components KO cells against the gWT cells. Data represent the mean and standard error of a minimum of three independent experiments. One clone is used for each genotype.
Figure Legend Snippet: Fitness of Ramos cells depends on the Igβ subunit of the BCR A schematic diagram showing the route for the generation of single‐ and multi‐BCR components KO from wild‐type (WT) Ramos B cells by the CRISPR/Cas9 method. Cell proliferation assay of WT and Ramos‐null cells using CytoTell™ UltraGreen. Null = HC/LC/Igα/Igβ tetra KO. A diagram depicting the growth competition assay. WT Ramos cells were retrovirally transduced with pMIG empty vector (EV) to create GFP‐labeled WT cells (gWT). BCR component KO cells were mixed together with gWT Ramos cells at about 1:1 ratio at day 0, and the relative amount of GFP − BCR components KO cells was then measured by flow cytometry at different time points. The expression levels of IgM‐BCR and IgD‐BCR on the surface of WT and Ramos‐null cells were determined by flow cytometry. Growth competition between Ramos‐null and gWT cells. The competition between GFP − WT and gWT cells serves as a control. The data represent the mean and standard error of a minimum of three independent experiments. The expression levels of IgM‐BCR and IgD‐BCR on the surface of WT and BCR component KO Ramos cells were determined by flow cytometry. Growth competition of the BCR components KO cells against the gWT cells. Data represent the mean and standard error of a minimum of three independent experiments. One clone is used for each genotype.

Techniques Used: CRISPR, Proliferation Assay, Competitive Binding Assay, Transduction, Plasmid Preparation, Labeling, Flow Cytometry, Cytometry, Expressing

1‐ PLA analysis of the Igβ: CD 19 proximity on splenic B cells from a tamoxifen treated B1‐8f/Δ mb1Cre ERT 2 mouse Representative microscopic image showing splenic B cells from a tamoxifen treated B1‐8f/Δ mb1CreERT2 mouse. The different colors indicate anti‐kappa LC staining (green), DAPI staining (blue), and the PLA signals (red). Scale bar: 5 μm. For PLA signal quantification, the image in (A) was segmented in silico to a BCR + (green) and BCR − (gray) cell population. Left panel shows a representative flow cytometry analysis to determine the percentage of BCR + cells (IgM + IgD + ) and BCR − cells (IgM − IgD − ) of B cells from the same spleen. Right panel shows the percentage of BCR + and BCR − cells after in silico segmentation. It also shows that the percentage of BCR + and BCR − cells of splenic cells from B1‐8f/Δ mb1CreERT2 mouse determined by in silico segmentation is as expected. Data represent the mean and the standard error of six experiments with each of them counting 50–200 cells. Quantification of the PLA signals in BCR + and BCR − cells on the segmented images. Data represent the mean and the standard error of six experiments with each of them counting 50–200 cells. P ‐values were calculated by Wilcoxon signed rank test.
Figure Legend Snippet: 1‐ PLA analysis of the Igβ: CD 19 proximity on splenic B cells from a tamoxifen treated B1‐8f/Δ mb1Cre ERT 2 mouse Representative microscopic image showing splenic B cells from a tamoxifen treated B1‐8f/Δ mb1CreERT2 mouse. The different colors indicate anti‐kappa LC staining (green), DAPI staining (blue), and the PLA signals (red). Scale bar: 5 μm. For PLA signal quantification, the image in (A) was segmented in silico to a BCR + (green) and BCR − (gray) cell population. Left panel shows a representative flow cytometry analysis to determine the percentage of BCR + cells (IgM + IgD + ) and BCR − cells (IgM − IgD − ) of B cells from the same spleen. Right panel shows the percentage of BCR + and BCR − cells after in silico segmentation. It also shows that the percentage of BCR + and BCR − cells of splenic cells from B1‐8f/Δ mb1CreERT2 mouse determined by in silico segmentation is as expected. Data represent the mean and the standard error of six experiments with each of them counting 50–200 cells. Quantification of the PLA signals in BCR + and BCR − cells on the segmented images. Data represent the mean and the standard error of six experiments with each of them counting 50–200 cells. P ‐values were calculated by Wilcoxon signed rank test.

Techniques Used: Proximity Ligation Assay, Staining, In Silico, Flow Cytometry, Cytometry

8) Product Images from "Blimp-1–dependent and –independent natural antibody production by B-1 and B-1–derived plasma cells"

Article Title: Blimp-1–dependent and –independent natural antibody production by B-1 and B-1–derived plasma cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20161122

Phenotypic B-1 cells and PCs secrete natural IgM predominantly in the spleen and BM. (A) Concentration ± SD (µg/ml) of IgM, IgG3, and IgA in serum of germ-free and SPF-maintained Swiss Webster mice ( n = 8–15) measured by ELISA. (B) Number ± SD of IgM ASCs as determined by ELISPOT ( n = 7 for germ-free mice and n = 4 for SPF mice). Results are combined (A) or representative (B) of two independent experiments. (C, left) Number ± SD of IgM ASCs in multiple tissues of wild-type neonatal chimeras as measured by ELISPOT ( n = 3–6). IgM a and IgM b spots were added for each mouse. (C, right) Representative ELISPOT demonstrating IgM secretion by spleen and BM cells of C57BL/6 control mice and mice lacking secretory IgM (µs−/− mice; n = 3). Numbers indicate total input cells. Representative of two independent experiments. (D) Gating strategy for CD19 + IgM +/neg cells after selecting “nondump,” CD43 + , IgD lo/neg cells. Values shown in all FACS plots represent percentage within parent population. (E) Frequency ± SD of IgM ASCs among live, nondump, CD43 + , IgD lo/neg cells in BM ( n = 2 for CD19 neg IgM + ; n = 5 for the others) and spleen ( n = 6–7 for IgM + ; n = 2–3 for IgM neg ). The dashed lines indicate limit of detection. Results are representative of two independent experiments. (F) Representative FACS histograms of CD138 expression among BM CD19 + IgM + (B-1 cells) and CD19 neg IgM + (PCs). (G) Contribution of B-1 cells and PCs to total IgM ASCs in indicated tissues was determined by multiplying the percentage of IgM ASCs in each population by the frequency of that population in the tissue ( n = 9). Results are combined from three to four independent experiments. Statistics in A and B were done using an unpaired Student’s t test (****, P
Figure Legend Snippet: Phenotypic B-1 cells and PCs secrete natural IgM predominantly in the spleen and BM. (A) Concentration ± SD (µg/ml) of IgM, IgG3, and IgA in serum of germ-free and SPF-maintained Swiss Webster mice ( n = 8–15) measured by ELISA. (B) Number ± SD of IgM ASCs as determined by ELISPOT ( n = 7 for germ-free mice and n = 4 for SPF mice). Results are combined (A) or representative (B) of two independent experiments. (C, left) Number ± SD of IgM ASCs in multiple tissues of wild-type neonatal chimeras as measured by ELISPOT ( n = 3–6). IgM a and IgM b spots were added for each mouse. (C, right) Representative ELISPOT demonstrating IgM secretion by spleen and BM cells of C57BL/6 control mice and mice lacking secretory IgM (µs−/− mice; n = 3). Numbers indicate total input cells. Representative of two independent experiments. (D) Gating strategy for CD19 + IgM +/neg cells after selecting “nondump,” CD43 + , IgD lo/neg cells. Values shown in all FACS plots represent percentage within parent population. (E) Frequency ± SD of IgM ASCs among live, nondump, CD43 + , IgD lo/neg cells in BM ( n = 2 for CD19 neg IgM + ; n = 5 for the others) and spleen ( n = 6–7 for IgM + ; n = 2–3 for IgM neg ). The dashed lines indicate limit of detection. Results are representative of two independent experiments. (F) Representative FACS histograms of CD138 expression among BM CD19 + IgM + (B-1 cells) and CD19 neg IgM + (PCs). (G) Contribution of B-1 cells and PCs to total IgM ASCs in indicated tissues was determined by multiplying the percentage of IgM ASCs in each population by the frequency of that population in the tissue ( n = 9). Results are combined from three to four independent experiments. Statistics in A and B were done using an unpaired Student’s t test (****, P

Techniques Used: Concentration Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, FACS, Expressing

9) Product Images from "Increased CD40 ligation and reduced BCR signalling leads to higher IL-10 production in B-cells from tolerant kidney transplant patients"

Article Title: Increased CD40 ligation and reduced BCR signalling leads to higher IL-10 production in B-cells from tolerant kidney transplant patients

Journal: Transplantation

doi: 10.1097/TP.0000000000001341

B-cells from tolerant recipients display a reduced ERK signalling after BCR activation. A) A phospho-flow B-cell panel was designed to identify p-ERK in B-cell subsets within PBMCs. B) A time course of p-ERK was measured in PBMCs after BCR-activation [anti-IgM (20μg/ml)/anti-IgG (20μg/ml)] during 0, 10 and 30min at 37°C, using PMA (0.1μM) for 10min as a positive control. C) Δp-ERK was defined as the difference between the p-ERK MFI from a non-activated and a 10min BCR-activated sample. D) The BCR signalling was measured in 1x10 6 PBMCs from healthy control (HC=9) and tolerant (Tol=9) patients after BCR-activation or after E) PMA (0.1μM) for 10min. F) IL-10 production after BCR/CD40 activation was measured by adding 0.5x10 5 non-transfected or CD40L-transfected L-cells in 0.5x10 6 non-activated or BCR-activated B-cell subsets from healthy leukocytes retained in filtering cones after 3 days of culture by intracellular staining. G) Levels of IL-10 were measured in the supernatants of 10x10 4 sorted transitional B-cells from 3 HC and 3 Tol recipients activated with 1x10 3 non-transfected or CD40L-transfected L-cells, with or without BCR activation for 3 days. H) IL-2 and IL-4 production from isolated CD4 + T-cells of HC=6 and Tol=7 were measured after 48h of activation with CD3/CD28 beads (1:2 ratio). Detection limit for IL-2 and IL-4 were 2.6 and 4.9pg/ml, respectively. I) Levels of IgG1 were measured in the sera from HC and Tol patients. Mann-Whitney test and two-way RM ANOVA test with a Sidak’s multiple comparisons test were used, **** P
Figure Legend Snippet: B-cells from tolerant recipients display a reduced ERK signalling after BCR activation. A) A phospho-flow B-cell panel was designed to identify p-ERK in B-cell subsets within PBMCs. B) A time course of p-ERK was measured in PBMCs after BCR-activation [anti-IgM (20μg/ml)/anti-IgG (20μg/ml)] during 0, 10 and 30min at 37°C, using PMA (0.1μM) for 10min as a positive control. C) Δp-ERK was defined as the difference between the p-ERK MFI from a non-activated and a 10min BCR-activated sample. D) The BCR signalling was measured in 1x10 6 PBMCs from healthy control (HC=9) and tolerant (Tol=9) patients after BCR-activation or after E) PMA (0.1μM) for 10min. F) IL-10 production after BCR/CD40 activation was measured by adding 0.5x10 5 non-transfected or CD40L-transfected L-cells in 0.5x10 6 non-activated or BCR-activated B-cell subsets from healthy leukocytes retained in filtering cones after 3 days of culture by intracellular staining. G) Levels of IL-10 were measured in the supernatants of 10x10 4 sorted transitional B-cells from 3 HC and 3 Tol recipients activated with 1x10 3 non-transfected or CD40L-transfected L-cells, with or without BCR activation for 3 days. H) IL-2 and IL-4 production from isolated CD4 + T-cells of HC=6 and Tol=7 were measured after 48h of activation with CD3/CD28 beads (1:2 ratio). Detection limit for IL-2 and IL-4 were 2.6 and 4.9pg/ml, respectively. I) Levels of IgG1 were measured in the sera from HC and Tol patients. Mann-Whitney test and two-way RM ANOVA test with a Sidak’s multiple comparisons test were used, **** P

Techniques Used: Activation Assay, Flow Cytometry, Positive Control, Transfection, Staining, Isolation, MANN-WHITNEY

10) Product Images from "Molecular requirements of the B‐cell antigen receptor for sensing monovalent antigens"

Article Title: Molecular requirements of the B‐cell antigen receptor for sensing monovalent antigens

Journal: The EMBO Journal

doi: 10.15252/embj.201694177

Lyn is indispensable for the opening and signalling of the BCR upon monovalent antigen binding Calcium flux measured by FACScan for splenic B cells isolated from Lyn‐deficient B1‐8 transgenic mice after stimulation with (A) NIP15‐BSA (30 pM) or 1NIP‐pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells. Representative microscopic images showing Fab‐PLA results monitoring the BCR proximity for the IgM‐BCR (upper) and the IgD‐BCR (lower) on untreated or treated B1‐8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. Quantified Fab‐PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample; P ‐values were calculated by Kruskal–Wallis one‐way ANOVA. Data information: Data are representative of at least three independent experiments.
Figure Legend Snippet: Lyn is indispensable for the opening and signalling of the BCR upon monovalent antigen binding Calcium flux measured by FACScan for splenic B cells isolated from Lyn‐deficient B1‐8 transgenic mice after stimulation with (A) NIP15‐BSA (30 pM) or 1NIP‐pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells. Representative microscopic images showing Fab‐PLA results monitoring the BCR proximity for the IgM‐BCR (upper) and the IgD‐BCR (lower) on untreated or treated B1‐8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. Quantified Fab‐PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample; P ‐values were calculated by Kruskal–Wallis one‐way ANOVA. Data information: Data are representative of at least three independent experiments.

Techniques Used: Binding Assay, Isolation, Transgenic Assay, Mouse Assay, Proximity Ligation Assay, Staining

The kinase activity of Lyn is crucial for monovalent antigen‐induced calcium response and BCR opening Calcium flux measured by FACScan for splenic B cells isolated from B1‐8 transgenic mice after the stimulation with NIP15‐BSA (30 pM), 1NIP‐pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM) after 45‐min incubation with 1 mM PP2. Arrows indicate the addition of the stimuli to the cells. Representative microscopic images showing Fab‐PLA results monitoring the BCR proximity for IgM‐BCR and IgD‐BCR on untreated or treated cells. PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. Quantified PLA results presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. P ‐values were calculated by Kruskal–Wallis one‐way analysis of variance (ANOVA). Data information: Data are representative of three independent experiments.
Figure Legend Snippet: The kinase activity of Lyn is crucial for monovalent antigen‐induced calcium response and BCR opening Calcium flux measured by FACScan for splenic B cells isolated from B1‐8 transgenic mice after the stimulation with NIP15‐BSA (30 pM), 1NIP‐pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM) after 45‐min incubation with 1 mM PP2. Arrows indicate the addition of the stimuli to the cells. Representative microscopic images showing Fab‐PLA results monitoring the BCR proximity for IgM‐BCR and IgD‐BCR on untreated or treated cells. PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. Quantified PLA results presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. P ‐values were calculated by Kruskal–Wallis one‐way analysis of variance (ANOVA). Data information: Data are representative of three independent experiments.

Techniques Used: Activity Assay, Isolation, Transgenic Assay, Mouse Assay, Incubation, Proximity Ligation Assay, Staining

Monovalent antigen binding opens BCR oligomers and induces a calcium flux in splenic B cells Calcium flux measured by FACScan for splenic B cells isolated from B1‐8 transgenic mice after stimulation with (A) NIP15‐BSA (30 pM) or 1NIP‐pep (see Fig EV1 , 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows. Representative microscopic images showing Fab‐PLA results measuring the BCR proximity for the IgM‐BCR (upper) and the IgD‐BCR (lower) on untreated or treated B1‐8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. The Fab‐PLA results are quantified by BlobFinder software and presented as box plots. The median values are highlighted as thick lines, and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from the treated samples were compared with data from the resting cells; P ‐values were calculated by Kruskal–Wallis one‐way analysis of variance (ANOVA). Data information: Data are representative of at least three independent experiments.
Figure Legend Snippet: Monovalent antigen binding opens BCR oligomers and induces a calcium flux in splenic B cells Calcium flux measured by FACScan for splenic B cells isolated from B1‐8 transgenic mice after stimulation with (A) NIP15‐BSA (30 pM) or 1NIP‐pep (see Fig EV1 , 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows. Representative microscopic images showing Fab‐PLA results measuring the BCR proximity for the IgM‐BCR (upper) and the IgD‐BCR (lower) on untreated or treated B1‐8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. The Fab‐PLA results are quantified by BlobFinder software and presented as box plots. The median values are highlighted as thick lines, and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from the treated samples were compared with data from the resting cells; P ‐values were calculated by Kruskal–Wallis one‐way analysis of variance (ANOVA). Data information: Data are representative of at least three independent experiments.

Techniques Used: Binding Assay, Isolation, Transgenic Assay, Mouse Assay, Proximity Ligation Assay, Staining, Software

11) Product Images from "A new branched proximity hybridization assay for the quantification of nanoscale protein–protein proximity"

Article Title: A new branched proximity hybridization assay for the quantification of nanoscale protein–protein proximity

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3000569

The bPHA works in a mixed cell population. (A) Schematic diagram showing the route map for generating GFP-μm-expressing IgM or IgD KO cells from WT Ramos B cells by CRSPR/Cas9 method. (B) Flow cytometry results showing that the subpopulations of the mixed cells can be identified by gating for the expression of GFP and anti-IgD staining. (C and D) TD05+:TD05− (C) or Enh+:Enh− (D) bPHA results were measured by flow cytometry and analyzed by the gating strategy shown in (B). Data represent three independent experiments. bPHA, branched proximity hybridization assay; Enh, Enhancer; GFP, Green fluorescent protein; IgD, immunoglobulin D; IgM, immunoglobulin M; KO, knock-out; PE, phycoerythrin; WT, wild-type.
Figure Legend Snippet: The bPHA works in a mixed cell population. (A) Schematic diagram showing the route map for generating GFP-μm-expressing IgM or IgD KO cells from WT Ramos B cells by CRSPR/Cas9 method. (B) Flow cytometry results showing that the subpopulations of the mixed cells can be identified by gating for the expression of GFP and anti-IgD staining. (C and D) TD05+:TD05− (C) or Enh+:Enh− (D) bPHA results were measured by flow cytometry and analyzed by the gating strategy shown in (B). Data represent three independent experiments. bPHA, branched proximity hybridization assay; Enh, Enhancer; GFP, Green fluorescent protein; IgD, immunoglobulin D; IgM, immunoglobulin M; KO, knock-out; PE, phycoerythrin; WT, wild-type.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Hybridization, Knock-Out

The bPHA confirms the rearrangement of BCR upon stimulation. (A) TD05+:TD05−, TD05+:Enh−, and Enh+:Enh− bPHA signals were measured by flow cytometry for resting and stimulated IgM-KO GFP-μm-expressing Ramos cells. The stimulated cells without the corresponding target binding probes served as control. Data represent four independent experiments. (B) Schematic diagrams showing that on the surface of IgM-KO GFP-μm-expressing Ramos cells surface, upon stimulation, IgD-BCR and GFP-μm mix together, producing positive TD05+:Enh− bPHA signal. BCR, B cell antigen receptor; bPHA, branched proximity hybridization assay; Enh, Enhancer; IgD, immunoglobulin D; IgM, immunoglobulin M; KO, knock-out.
Figure Legend Snippet: The bPHA confirms the rearrangement of BCR upon stimulation. (A) TD05+:TD05−, TD05+:Enh−, and Enh+:Enh− bPHA signals were measured by flow cytometry for resting and stimulated IgM-KO GFP-μm-expressing Ramos cells. The stimulated cells without the corresponding target binding probes served as control. Data represent four independent experiments. (B) Schematic diagrams showing that on the surface of IgM-KO GFP-μm-expressing Ramos cells surface, upon stimulation, IgD-BCR and GFP-μm mix together, producing positive TD05+:Enh− bPHA signal. BCR, B cell antigen receptor; bPHA, branched proximity hybridization assay; Enh, Enhancer; IgD, immunoglobulin D; IgM, immunoglobulin M; KO, knock-out.

Techniques Used: Flow Cytometry, Cytometry, Expressing, Binding Assay, Hybridization, Knock-Out

The bPHA uncovers class-specific kinetics of Syk recruitment to BCR. (A) Schematic diagrams showing that upon stimulation, Syk can be recruited to CD79a. The proximity between CD79a and Syk can be measured by bPHA using oligo-coupled anti-CD79a and anti-Syk antibodies. (B) Anti-CD79a+:anti-Syk− bPHA signals were measured by flow cytometry for resting and anti-IgD- or anti-IgM-stimulated splenic B cells. Cells without the corresponding target binding probes served as controls for both the resting and stimulated cells. Data represent three independent experiments. BCR, B cell antigen receptor; bPHA, branched proximity hybridization assay; IgD, immunoglobulin D; IgM, immunoglobulin M; Syk, spleen tyrosine kinase.
Figure Legend Snippet: The bPHA uncovers class-specific kinetics of Syk recruitment to BCR. (A) Schematic diagrams showing that upon stimulation, Syk can be recruited to CD79a. The proximity between CD79a and Syk can be measured by bPHA using oligo-coupled anti-CD79a and anti-Syk antibodies. (B) Anti-CD79a+:anti-Syk− bPHA signals were measured by flow cytometry for resting and anti-IgD- or anti-IgM-stimulated splenic B cells. Cells without the corresponding target binding probes served as controls for both the resting and stimulated cells. Data represent three independent experiments. BCR, B cell antigen receptor; bPHA, branched proximity hybridization assay; IgD, immunoglobulin D; IgM, immunoglobulin M; Syk, spleen tyrosine kinase.

Techniques Used: Flow Cytometry, Cytometry, Binding Assay, Hybridization

12) Product Images from "TACI Isoforms Regulate Ligand Binding and Receptor Function"

Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02125

TACI isoforms expression in activated B cells. TACI isoform expression in non-permeabilized (A) and permeabilized (B) peripheral blood B cells from normal donors, cultured for 4 days in the presence or absence of ODN, CD40L/IL21, or anti-IgM. Data shows the mean ± SEM of 4 independent experiments. (C,D) TACI isoform expression was analyzed in B cell subpopulations isolated from freshly-isolated tonsils ( n = 4): Naïve (IgD+CD27-), Transitional (Tr) (IgM+CD38+), Marginal Zone (MZ) (IgD+CD27+), Switched Memory (SwMe) (IgD-CD27+), and Plasmablasts (PB) (IgD-CD27 hi CD38 hi ). Non permeabilized (C) and permeabilized cells (D) were examined. Data shows the mean ± SEM. * p
Figure Legend Snippet: TACI isoforms expression in activated B cells. TACI isoform expression in non-permeabilized (A) and permeabilized (B) peripheral blood B cells from normal donors, cultured for 4 days in the presence or absence of ODN, CD40L/IL21, or anti-IgM. Data shows the mean ± SEM of 4 independent experiments. (C,D) TACI isoform expression was analyzed in B cell subpopulations isolated from freshly-isolated tonsils ( n = 4): Naïve (IgD+CD27-), Transitional (Tr) (IgM+CD38+), Marginal Zone (MZ) (IgD+CD27+), Switched Memory (SwMe) (IgD-CD27+), and Plasmablasts (PB) (IgD-CD27 hi CD38 hi ). Non permeabilized (C) and permeabilized cells (D) were examined. Data shows the mean ± SEM. * p

Techniques Used: Expressing, Cell Culture, Isolation

13) Product Images from "Expression and function of the TL1A/DR3 axis in chronic lymphocytic leukemia"

Article Title: Expression and function of the TL1A/DR3 axis in chronic lymphocytic leukemia

Journal: Oncotarget

doi:

DR3 surface expression in CLL cells A. Representative example of DR3 flow cytometry analysis. Dashed line: isotypic control; regular line: anti-DR3 signal. B. Time course analysis of DR3 expression in unstimulated and anti-IgM-stimulated CLL cells ( n = 8). C. Surface DR3 expression in unstimulated and anti-IgM-stimulated CLL cells ( n = 36). Data are expressed as DR3-expression median fluorescence intensity (MFI) divided by isotype-matched control (relative median fluorescence intensity = RMFI). Comparison between unstimulated and anti-IgM-stimulated CLL cells was performed by the two-sample Wilcoxon signed rank sum test. D. Comparison of DR3 expression between B cells from CLL samples ( n = 36) and age-matched healthy donors ( n = 10). Lines represent median values. Comparison was performed using the Mann-Whitney test. ns = not significant. E. Western blot analysis of cell lysates from purified CLL cells ( n = 4), in unstimulated and anti-IgM stimulated conditions. Level of DR3 induction after anti-IgM stimulation is reported as fold change. F. Representative example of DR3 immunofluorescence analysis in CLL lymph-node tissue sections ( n = 2). Panel A: pseudocolour image of DR3 (200×); Panel B: pseudocolour image of CD23 (200×); Panel C: merged pseudocolour image of CD23 (green), DR3 (red) and DNA (blu) (200×).
Figure Legend Snippet: DR3 surface expression in CLL cells A. Representative example of DR3 flow cytometry analysis. Dashed line: isotypic control; regular line: anti-DR3 signal. B. Time course analysis of DR3 expression in unstimulated and anti-IgM-stimulated CLL cells ( n = 8). C. Surface DR3 expression in unstimulated and anti-IgM-stimulated CLL cells ( n = 36). Data are expressed as DR3-expression median fluorescence intensity (MFI) divided by isotype-matched control (relative median fluorescence intensity = RMFI). Comparison between unstimulated and anti-IgM-stimulated CLL cells was performed by the two-sample Wilcoxon signed rank sum test. D. Comparison of DR3 expression between B cells from CLL samples ( n = 36) and age-matched healthy donors ( n = 10). Lines represent median values. Comparison was performed using the Mann-Whitney test. ns = not significant. E. Western blot analysis of cell lysates from purified CLL cells ( n = 4), in unstimulated and anti-IgM stimulated conditions. Level of DR3 induction after anti-IgM stimulation is reported as fold change. F. Representative example of DR3 immunofluorescence analysis in CLL lymph-node tissue sections ( n = 2). Panel A: pseudocolour image of DR3 (200×); Panel B: pseudocolour image of CD23 (200×); Panel C: merged pseudocolour image of CD23 (green), DR3 (red) and DNA (blu) (200×).

Techniques Used: Expressing, Flow Cytometry, Cytometry, Fluorescence, MANN-WHITNEY, Western Blot, Purification, Immunofluorescence

14) Product Images from "IL-17 augments B cell activation in ocular surface autoimmunity"

Article Title: IL-17 augments B cell activation in ocular surface autoimmunity

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

doi: 10.4049/jimmunol.1502641

B cell proliferation induced by IgM/CD40 stimulation is enhanced by co-stimulation with IL-17 only in autoimmune B cells (A) anti-IgM/anti-CD40–stimulated B cells were cultured at the bottom of the transwell, and anti-CD3-stimulated T effector cells were cultured in the insert (top) in the presence or absence of anti-IL-17A antibody. Flow cytometric analysis showing Ki67 staining of purified B cells from dry eye (DED) mice. (B) Naive or DED B cells were stimulated with anti-IgM/anti-CD40 and with or without recombinant IL-17A. Flow cytometric analysis showing Ki67 staining of purified B cells from DED and naïve mice after 48 hrs stimulation. B cell proliferation is shown as fold change (B cell+ anti-IgM and anti-CD40 =1). All results are representative of three independent experiments. (mean ± SEM; n=4–6 mice/group; triplicate cultures). Student’s t test **, P
Figure Legend Snippet: B cell proliferation induced by IgM/CD40 stimulation is enhanced by co-stimulation with IL-17 only in autoimmune B cells (A) anti-IgM/anti-CD40–stimulated B cells were cultured at the bottom of the transwell, and anti-CD3-stimulated T effector cells were cultured in the insert (top) in the presence or absence of anti-IL-17A antibody. Flow cytometric analysis showing Ki67 staining of purified B cells from dry eye (DED) mice. (B) Naive or DED B cells were stimulated with anti-IgM/anti-CD40 and with or without recombinant IL-17A. Flow cytometric analysis showing Ki67 staining of purified B cells from DED and naïve mice after 48 hrs stimulation. B cell proliferation is shown as fold change (B cell+ anti-IgM and anti-CD40 =1). All results are representative of three independent experiments. (mean ± SEM; n=4–6 mice/group; triplicate cultures). Student’s t test **, P

Techniques Used: Cell Culture, Flow Cytometry, Staining, Purification, Mouse Assay, Recombinant

Activated B cells show enhanced IL-17 receptor expression (A) Immunohistochemistry was performed on submandibular lymph nodes of naïve and dry eye (DED) mice (21 days after DED induction) to visualize Ki67 (proliferating cells, green), IgM (B cells, red), and IL-17RA (receptor expression, blue) expression. Data are representative of 4–6 mice examined. Images are 10x magnification and 20x magnification for magnified view of areas in white box. (B) Flow cytomteric analysis showing IL-17RA+ CD19+ cells after stimulating purified B cells with medium alone or in the presence or absence of anti-IgM, anti-CD40, and IL-17A for 48 hrs. (mean ± SEM; n=4 mice/group; triplicate cultures). Two-way ANOVA. **, P
Figure Legend Snippet: Activated B cells show enhanced IL-17 receptor expression (A) Immunohistochemistry was performed on submandibular lymph nodes of naïve and dry eye (DED) mice (21 days after DED induction) to visualize Ki67 (proliferating cells, green), IgM (B cells, red), and IL-17RA (receptor expression, blue) expression. Data are representative of 4–6 mice examined. Images are 10x magnification and 20x magnification for magnified view of areas in white box. (B) Flow cytomteric analysis showing IL-17RA+ CD19+ cells after stimulating purified B cells with medium alone or in the presence or absence of anti-IgM, anti-CD40, and IL-17A for 48 hrs. (mean ± SEM; n=4 mice/group; triplicate cultures). Two-way ANOVA. **, P

Techniques Used: Expressing, Immunohistochemistry, Mouse Assay, Flow Cytometry, Purification

15) Product Images from "GIMAP1 is essential for the survival of naïve and activated B cells in vivo"

Article Title: GIMAP1 is essential for the survival of naïve and activated B cells in vivo

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

doi: 10.4049/jimmunol.1501582

GIMAP1 is essential for germinal center B cell responses (A) Conditional deletion of Gimap1 in germinal center B cells. PCR analysis of Gimap1 and Gimap8 in germinal center B cells FACS-sorted from immunized Gimap1 fl/fl AicdaCre + and Gimap1 fl/fl mice. Lane 1 = 100bp DNA ladder; lane 2 = follicular B cells from control Gimap1 fl/fl mice; lane 3 = germinal center B cells from control Gimap1 fl/fl mice; lane 4 = follicular B cells from Gimap1 fl/fl AicdaCre + mice; lane 5 = germinal center B cells from Gimap1 fl/fl AicdaCre + mice; lane 6 = H 2 O control. (B) Facsplots showing NIP-binding IgG1-switched B cells in Gimap1 fl/fl AicdaCre + and Gimap1 fl/fl mice on day 7 p.i. (C) Enumeration of NIP-binding IgG1-switched (B220 +ve IgM −ve IgD −ve ) B cells on day 7 p.i. in Gimap1 fl/fl (●) and Gimap1 fl/fl AicdaCre + (□). Results show the number of cells per spleen for individual mice with the mean ± S.D. (D) Titres of NP23-binding (low affinity) and NP2-binding (high affinity) IgG1 and IgM antibodies on days 7 and 14 after primary immunization. Each symbol represents an individual mouse ( Gimap1 fl/fl (●) and Gimap1 fl/fl AicdaCre + (□) with the mean ± S.D. shown. (E) The frequency of NP-specific IgG1 and IgM ASC from Gimap1 fl/f (●) and Gimap1 fl/fl AicdaCre + (□) mice 14 days p.i. as determined by ELISPOT. Each symbol represents an individual mouse with the mean ± S.D. shown. Differences were examined using an unpaired Student’s t test and only significant differences are marked (* p
Figure Legend Snippet: GIMAP1 is essential for germinal center B cell responses (A) Conditional deletion of Gimap1 in germinal center B cells. PCR analysis of Gimap1 and Gimap8 in germinal center B cells FACS-sorted from immunized Gimap1 fl/fl AicdaCre + and Gimap1 fl/fl mice. Lane 1 = 100bp DNA ladder; lane 2 = follicular B cells from control Gimap1 fl/fl mice; lane 3 = germinal center B cells from control Gimap1 fl/fl mice; lane 4 = follicular B cells from Gimap1 fl/fl AicdaCre + mice; lane 5 = germinal center B cells from Gimap1 fl/fl AicdaCre + mice; lane 6 = H 2 O control. (B) Facsplots showing NIP-binding IgG1-switched B cells in Gimap1 fl/fl AicdaCre + and Gimap1 fl/fl mice on day 7 p.i. (C) Enumeration of NIP-binding IgG1-switched (B220 +ve IgM −ve IgD −ve ) B cells on day 7 p.i. in Gimap1 fl/fl (●) and Gimap1 fl/fl AicdaCre + (□). Results show the number of cells per spleen for individual mice with the mean ± S.D. (D) Titres of NP23-binding (low affinity) and NP2-binding (high affinity) IgG1 and IgM antibodies on days 7 and 14 after primary immunization. Each symbol represents an individual mouse ( Gimap1 fl/fl (●) and Gimap1 fl/fl AicdaCre + (□) with the mean ± S.D. shown. (E) The frequency of NP-specific IgG1 and IgM ASC from Gimap1 fl/f (●) and Gimap1 fl/fl AicdaCre + (□) mice 14 days p.i. as determined by ELISPOT. Each symbol represents an individual mouse with the mean ± S.D. shown. Differences were examined using an unpaired Student’s t test and only significant differences are marked (* p

Techniques Used: Polymerase Chain Reaction, FACS, Mouse Assay, Binding Assay, Enzyme-linked Immunospot

Failure to establish B cell memory in the absence of GIMAP1 Secondary responses in Gimap1 fl/fl AicdaCre + (□) and control Gimap1 fl/f (●) mice: each symbol represents an individual mouse with mean ± S.D. also shown. (A) NIP-binding, IgG1-switched splenic B cells enumerated using flow cytometric analysis. (B) Titres of NP23-binding (total affinity) and NP2-binding (high affinity) IgG1 and IgM antibodies on day 7 of secondary immunization. (C) The frequency of NP-specific IgG1 and IgM ASC from Gimap1 fl/fl (●) and Gimap1 fl/fl AicdaCre + (□) mice 7 days p.i. as determined by ELISPOT is shown. Each symbol represents an individual mouse with the mean ± S.D. shown. Differences were examined using an unpaired Student’s t test and only significant differences are marked (* p
Figure Legend Snippet: Failure to establish B cell memory in the absence of GIMAP1 Secondary responses in Gimap1 fl/fl AicdaCre + (□) and control Gimap1 fl/f (●) mice: each symbol represents an individual mouse with mean ± S.D. also shown. (A) NIP-binding, IgG1-switched splenic B cells enumerated using flow cytometric analysis. (B) Titres of NP23-binding (total affinity) and NP2-binding (high affinity) IgG1 and IgM antibodies on day 7 of secondary immunization. (C) The frequency of NP-specific IgG1 and IgM ASC from Gimap1 fl/fl (●) and Gimap1 fl/fl AicdaCre + (□) mice 7 days p.i. as determined by ELISPOT is shown. Each symbol represents an individual mouse with the mean ± S.D. shown. Differences were examined using an unpaired Student’s t test and only significant differences are marked (* p

Techniques Used: Mouse Assay, Binding Assay, Flow Cytometry, Enzyme-linked Immunospot

16) Product Images from "Molecular requirements of the B‐cell antigen receptor for sensing monovalent antigens"

Article Title: Molecular requirements of the B‐cell antigen receptor for sensing monovalent antigens

Journal: The EMBO Journal

doi: 10.15252/embj.201694177

Lyn is indispensable for the opening and signalling of the BCR upon monovalent antigen binding Calcium flux measured by FACScan for splenic B cells isolated from Lyn‐deficient B1‐8 transgenic mice after stimulation with (A) NIP15‐BSA (30 pM) or 1NIP‐pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells. Representative microscopic images showing Fab‐PLA results monitoring the BCR proximity for the IgM‐BCR (upper) and the IgD‐BCR (lower) on untreated or treated B1‐8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. Quantified Fab‐PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample; P ‐values were calculated by Kruskal–Wallis one‐way ANOVA. Data information: Data are representative of at least three independent experiments.
Figure Legend Snippet: Lyn is indispensable for the opening and signalling of the BCR upon monovalent antigen binding Calcium flux measured by FACScan for splenic B cells isolated from Lyn‐deficient B1‐8 transgenic mice after stimulation with (A) NIP15‐BSA (30 pM) or 1NIP‐pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells. Representative microscopic images showing Fab‐PLA results monitoring the BCR proximity for the IgM‐BCR (upper) and the IgD‐BCR (lower) on untreated or treated B1‐8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. Quantified Fab‐PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample; P ‐values were calculated by Kruskal–Wallis one‐way ANOVA. Data information: Data are representative of at least three independent experiments.

Techniques Used: Binding Assay, Isolation, Transgenic Assay, Mouse Assay, Proximity Ligation Assay, Staining

The kinase activity of Lyn is crucial for monovalent antigen‐induced calcium response and BCR opening Calcium flux measured by FACScan for splenic B cells isolated from B1‐8 transgenic mice after the stimulation with NIP15‐BSA (30 pM), 1NIP‐pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM) after 45‐min incubation with 1 mM PP2. Arrows indicate the addition of the stimuli to the cells. Representative microscopic images showing Fab‐PLA results monitoring the BCR proximity for IgM‐BCR and IgD‐BCR on untreated or treated cells. PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. Quantified PLA results presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. P ‐values were calculated by Kruskal–Wallis one‐way analysis of variance (ANOVA). Data information: Data are representative of three independent experiments.
Figure Legend Snippet: The kinase activity of Lyn is crucial for monovalent antigen‐induced calcium response and BCR opening Calcium flux measured by FACScan for splenic B cells isolated from B1‐8 transgenic mice after the stimulation with NIP15‐BSA (30 pM), 1NIP‐pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM) after 45‐min incubation with 1 mM PP2. Arrows indicate the addition of the stimuli to the cells. Representative microscopic images showing Fab‐PLA results monitoring the BCR proximity for IgM‐BCR and IgD‐BCR on untreated or treated cells. PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. Quantified PLA results presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. P ‐values were calculated by Kruskal–Wallis one‐way analysis of variance (ANOVA). Data information: Data are representative of three independent experiments.

Techniques Used: Activity Assay, Isolation, Transgenic Assay, Mouse Assay, Incubation, Proximity Ligation Assay, Staining

Monovalent antigen binding to IgM‐ BCR induces calcium signalling in a SLP ‐65 dependent manner Proximity between IgM‐BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1‐min stimulation with the indicated reagents, assayed by Fab‐PLA. Results are presented as representative microscopic images (A, B) and box plots after quantification (C, D). PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample, and P ‐values were calculated by Kruskal–Wallis one‐way ANOVA. Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15‐BSA (30 pM), 1NIP‐pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells. Data information: Data are representative of a minimum of three independent experiments.
Figure Legend Snippet: Monovalent antigen binding to IgM‐ BCR induces calcium signalling in a SLP ‐65 dependent manner Proximity between IgM‐BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1‐min stimulation with the indicated reagents, assayed by Fab‐PLA. Results are presented as representative microscopic images (A, B) and box plots after quantification (C, D). PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample, and P ‐values were calculated by Kruskal–Wallis one‐way ANOVA. Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15‐BSA (30 pM), 1NIP‐pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells. Data information: Data are representative of a minimum of three independent experiments.

Techniques Used: Binding Assay, Proximity Ligation Assay, Staining

Monovalent antigen binding opens BCR oligomers and induces a calcium flux in splenic B cells Calcium flux measured by FACScan for splenic B cells isolated from B1‐8 transgenic mice after stimulation with (A) NIP15‐BSA (30 pM) or 1NIP‐pep (see Fig EV1 , 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows. Representative microscopic images showing Fab‐PLA results measuring the BCR proximity for the IgM‐BCR (upper) and the IgD‐BCR (lower) on untreated or treated B1‐8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. The Fab‐PLA results are quantified by BlobFinder software and presented as box plots. The median values are highlighted as thick lines, and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from the treated samples were compared with data from the resting cells; P ‐values were calculated by Kruskal–Wallis one‐way analysis of variance (ANOVA). Data information: Data are representative of at least three independent experiments.
Figure Legend Snippet: Monovalent antigen binding opens BCR oligomers and induces a calcium flux in splenic B cells Calcium flux measured by FACScan for splenic B cells isolated from B1‐8 transgenic mice after stimulation with (A) NIP15‐BSA (30 pM) or 1NIP‐pep (see Fig EV1 , 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows. Representative microscopic images showing Fab‐PLA results measuring the BCR proximity for the IgM‐BCR (upper) and the IgD‐BCR (lower) on untreated or treated B1‐8 splenic B cells. PLA signals are shown as red dots, and nuclei were visualized by DAPI staining. Scale bar: 5 μm. The Fab‐PLA results are quantified by BlobFinder software and presented as box plots. The median values are highlighted as thick lines, and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from the treated samples were compared with data from the resting cells; P ‐values were calculated by Kruskal–Wallis one‐way analysis of variance (ANOVA). Data information: Data are representative of at least three independent experiments.

Techniques Used: Binding Assay, Isolation, Transgenic Assay, Mouse Assay, Proximity Ligation Assay, Staining, Software

17) Product Images from "Resolution of skeletal muscle inflammation in mdx dystrophic mouse is accompanied by increased immunoglobulin and interferon-? production"

Article Title: Resolution of skeletal muscle inflammation in mdx dystrophic mouse is accompanied by increased immunoglobulin and interferon-? production

Journal: International Journal of Experimental Pathology

doi: 10.1046/j.1365-2613.2002.00221.x

In vitro immunoglobulin secretion in the supernatants of lymphocytes derived from axillary and brachial lymph nodes of 12 week mdx mice cultivated in presence of LPS (10 µg/mL). Panel (a) shows IgM, IgG and IgA secretion in C57BL/ 10 or mdx supernatants. Panel (b) shows antigen reactivity of lymphocyte supernatants. One-hundred µL of protein antigens (10 µg/mL) were used for coating wells of microtitre plates. Supernatant reactivity was assessed with anti-IgM antibodies by direct ELISA. Representative experiments are presented. Results are expressed as mean ± SD of five animals. Similar results were obtained in three separate experiments. □ C57BL/ 10, mdx. ** P
Figure Legend Snippet: In vitro immunoglobulin secretion in the supernatants of lymphocytes derived from axillary and brachial lymph nodes of 12 week mdx mice cultivated in presence of LPS (10 µg/mL). Panel (a) shows IgM, IgG and IgA secretion in C57BL/ 10 or mdx supernatants. Panel (b) shows antigen reactivity of lymphocyte supernatants. One-hundred µL of protein antigens (10 µg/mL) were used for coating wells of microtitre plates. Supernatant reactivity was assessed with anti-IgM antibodies by direct ELISA. Representative experiments are presented. Results are expressed as mean ± SD of five animals. Similar results were obtained in three separate experiments. □ C57BL/ 10, mdx. ** P

Techniques Used: In Vitro, Derivative Assay, Mouse Assay, Direct ELISA

Numbers of Ig-secreting cells in draining lymph nodes (a, d), spleen (b, e) and bone marrow (c, f) of control and mdx mice at 12 weeks (a, b, c) and 24 weeks (d, e, f). Each symbol represents one animal. • IgM, ♦ IgG and ▴ IgA for mdx and corresponding open symbols for control mice. Bars depict mean values.
Figure Legend Snippet: Numbers of Ig-secreting cells in draining lymph nodes (a, d), spleen (b, e) and bone marrow (c, f) of control and mdx mice at 12 weeks (a, b, c) and 24 weeks (d, e, f). Each symbol represents one animal. • IgM, ♦ IgG and ▴ IgA for mdx and corresponding open symbols for control mice. Bars depict mean values.

Techniques Used: Mouse Assay

18) Product Images from "IL-17 augments B cell activation in ocular surface autoimmunity"

Article Title: IL-17 augments B cell activation in ocular surface autoimmunity

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

doi: 10.4049/jimmunol.1502641

IL-17 directly promotes B cell class switching and plasma cell formation (A) Purified B cells from dry eye (DED) mice were cultured in medium or with anti-IgM, IFN-γ, or IL-17 for 48 hrs and then analyzed for their IgM/IgD expression. (B) Flow cytometric analysis showing B cells cultured as described in (A) were analyzed for their expression of syndecan-1 (CD138, a plasma cell marker) (mean ± SEM; n=6 mice/group; triplicate cultures). Data are representative of two independent experiments. Student’s t test *, P
Figure Legend Snippet: IL-17 directly promotes B cell class switching and plasma cell formation (A) Purified B cells from dry eye (DED) mice were cultured in medium or with anti-IgM, IFN-γ, or IL-17 for 48 hrs and then analyzed for their IgM/IgD expression. (B) Flow cytometric analysis showing B cells cultured as described in (A) were analyzed for their expression of syndecan-1 (CD138, a plasma cell marker) (mean ± SEM; n=6 mice/group; triplicate cultures). Data are representative of two independent experiments. Student’s t test *, P

Techniques Used: Purification, Mouse Assay, Cell Culture, Expressing, Flow Cytometry, Marker

19) Product Images from "IL-17 augments B cell activation in ocular surface autoimmunity"

Article Title: IL-17 augments B cell activation in ocular surface autoimmunity

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

doi: 10.4049/jimmunol.1502641

B cell proliferation induced by IgM/CD40 stimulation is enhanced by co-stimulation with IL-17 only in autoimmune B cells (A) anti-IgM/anti-CD40–stimulated B cells were cultured at the bottom of the transwell, and anti-CD3-stimulated T effector cells were cultured in the insert (top) in the presence or absence of anti-IL-17A antibody. Flow cytometric analysis showing Ki67 staining of purified B cells from dry eye (DED) mice. (B) Naive or DED B cells were stimulated with anti-IgM/anti-CD40 and with or without recombinant IL-17A. Flow cytometric analysis showing Ki67 staining of purified B cells from DED and naïve mice after 48 hrs stimulation. B cell proliferation is shown as fold change (B cell+ anti-IgM and anti-CD40 =1). All results are representative of three independent experiments. (mean ± SEM; n=4–6 mice/group; triplicate cultures). Student’s t test **, P
Figure Legend Snippet: B cell proliferation induced by IgM/CD40 stimulation is enhanced by co-stimulation with IL-17 only in autoimmune B cells (A) anti-IgM/anti-CD40–stimulated B cells were cultured at the bottom of the transwell, and anti-CD3-stimulated T effector cells were cultured in the insert (top) in the presence or absence of anti-IL-17A antibody. Flow cytometric analysis showing Ki67 staining of purified B cells from dry eye (DED) mice. (B) Naive or DED B cells were stimulated with anti-IgM/anti-CD40 and with or without recombinant IL-17A. Flow cytometric analysis showing Ki67 staining of purified B cells from DED and naïve mice after 48 hrs stimulation. B cell proliferation is shown as fold change (B cell+ anti-IgM and anti-CD40 =1). All results are representative of three independent experiments. (mean ± SEM; n=4–6 mice/group; triplicate cultures). Student’s t test **, P

Techniques Used: Cell Culture, Flow Cytometry, Staining, Purification, Mouse Assay, Recombinant

Activated B cells show enhanced IL-17 receptor expression (A) Immunohistochemistry was performed on submandibular lymph nodes of naïve and dry eye (DED) mice (21 days after DED induction) to visualize Ki67 (proliferating cells, green), IgM (B cells, red), and IL-17RA (receptor expression, blue) expression. Data are representative of 4–6 mice examined. Images are 10x magnification and 20x magnification for magnified view of areas in white box. (B) Flow cytomteric analysis showing IL-17RA+ CD19+ cells after stimulating purified B cells with medium alone or in the presence or absence of anti-IgM, anti-CD40, and IL-17A for 48 hrs. (mean ± SEM; n=4 mice/group; triplicate cultures). Two-way ANOVA. **, P
Figure Legend Snippet: Activated B cells show enhanced IL-17 receptor expression (A) Immunohistochemistry was performed on submandibular lymph nodes of naïve and dry eye (DED) mice (21 days after DED induction) to visualize Ki67 (proliferating cells, green), IgM (B cells, red), and IL-17RA (receptor expression, blue) expression. Data are representative of 4–6 mice examined. Images are 10x magnification and 20x magnification for magnified view of areas in white box. (B) Flow cytomteric analysis showing IL-17RA+ CD19+ cells after stimulating purified B cells with medium alone or in the presence or absence of anti-IgM, anti-CD40, and IL-17A for 48 hrs. (mean ± SEM; n=4 mice/group; triplicate cultures). Two-way ANOVA. **, P

Techniques Used: Expressing, Immunohistochemistry, Mouse Assay, Flow Cytometry, Purification

20) Product Images from "TACI Isoforms Regulate Ligand Binding and Receptor Function"

Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02125

TACI isoforms expression in activated B cells. TACI isoform expression in non-permeabilized (A) and permeabilized (B) peripheral blood B cells from normal donors, cultured for 4 days in the presence or absence of ODN, CD40L/IL21, or anti-IgM. Data shows the mean ± SEM of 4 independent experiments. (C,D) TACI isoform expression was analyzed in B cell subpopulations isolated from freshly-isolated tonsils ( n = 4): Naïve (IgD+CD27-), Transitional (Tr) (IgM+CD38+), Marginal Zone (MZ) (IgD+CD27+), Switched Memory (SwMe) (IgD-CD27+), and Plasmablasts (PB) (IgD-CD27 hi CD38 hi ). Non permeabilized (C) and permeabilized cells (D) were examined. Data shows the mean ± SEM. * p
Figure Legend Snippet: TACI isoforms expression in activated B cells. TACI isoform expression in non-permeabilized (A) and permeabilized (B) peripheral blood B cells from normal donors, cultured for 4 days in the presence or absence of ODN, CD40L/IL21, or anti-IgM. Data shows the mean ± SEM of 4 independent experiments. (C,D) TACI isoform expression was analyzed in B cell subpopulations isolated from freshly-isolated tonsils ( n = 4): Naïve (IgD+CD27-), Transitional (Tr) (IgM+CD38+), Marginal Zone (MZ) (IgD+CD27+), Switched Memory (SwMe) (IgD-CD27+), and Plasmablasts (PB) (IgD-CD27 hi CD38 hi ). Non permeabilized (C) and permeabilized cells (D) were examined. Data shows the mean ± SEM. * p

Techniques Used: Expressing, Cell Culture, Isolation

21) Product Images from "A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection"

Article Title: A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007179

Zp-V3 (but not Zp-P) binds NFATc1. (A) EMSA oligonucleotides were designed to encompass the regions of Zp-V3 and Zp-P from -155 to -127, or from -155 to -120 and radiolabeled with 32 P. The potential NFAT (in Zp-V3, not Zp-P) and AP1 binding sites (in both variants) are indicated. (B) EBV-negative BJAB cells were transfected with the Zp-V3 luciferase vector, and then treated 24 hours later with or without anti-IgM, in the presence or absence of cyclosporine A (1μM) as indicated. Luciferase activity was measured 24 hours after anti-IgM treatment and results were normalized to that of the Zp-V3 untreated condition. (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) Nuclear extracts prepared from BJAB cells, treated with or without anti-IgM for 30 minutes or 6 hours, were incubated with the radiolabeled Zp-P or Zp-V3 (-155 to -127) probes in an EMSA. A protein that binds only to the Zp-V3 probe is indicated by an arrow. (D) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. Cold competitor DNA containing either the Zp-P or Zp-V3 oligonucleotides, or the consensus binding sites for the transcription factors indicated, was added in some conditions. An arrow depicts bands representing NFAT binding. (E) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. In some conditions, antibodies against NFATc1 or C/EBPα were added to the nuclear extract (prior to the addition of the labeled probe) as shown. An arrow depicts bands representing NFAT binding.
Figure Legend Snippet: Zp-V3 (but not Zp-P) binds NFATc1. (A) EMSA oligonucleotides were designed to encompass the regions of Zp-V3 and Zp-P from -155 to -127, or from -155 to -120 and radiolabeled with 32 P. The potential NFAT (in Zp-V3, not Zp-P) and AP1 binding sites (in both variants) are indicated. (B) EBV-negative BJAB cells were transfected with the Zp-V3 luciferase vector, and then treated 24 hours later with or without anti-IgM, in the presence or absence of cyclosporine A (1μM) as indicated. Luciferase activity was measured 24 hours after anti-IgM treatment and results were normalized to that of the Zp-V3 untreated condition. (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) Nuclear extracts prepared from BJAB cells, treated with or without anti-IgM for 30 minutes or 6 hours, were incubated with the radiolabeled Zp-P or Zp-V3 (-155 to -127) probes in an EMSA. A protein that binds only to the Zp-V3 probe is indicated by an arrow. (D) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. Cold competitor DNA containing either the Zp-P or Zp-V3 oligonucleotides, or the consensus binding sites for the transcription factors indicated, was added in some conditions. An arrow depicts bands representing NFAT binding. (E) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. In some conditions, antibodies against NFATc1 or C/EBPα were added to the nuclear extract (prior to the addition of the labeled probe) as shown. An arrow depicts bands representing NFAT binding.

Techniques Used: Binding Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Incubation, Labeling

22) Product Images from "A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection"

Article Title: A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007179

Residue -141 in the Zp-V3 promoter is critical for response to BCR crosslinking. (A) The Z promoter sequences in the B95.8 (top) and M81 (bottom) EBV strains are shown. B95.8 is the prototype (Zp-P) while M81 has the 3 polymorphisms at -100, -106, and -141 that define Variant 3 (Zp-V3). There are 4 additional basepair differences between the two Zp sequences, located at -274, -365, -460, and -525 (relative to the transcriptional start site), highlighted in gray. (B) EBV-negative BJAB cells were transfected with wildtype or mutant Zp-V3 luciferase constructs (named to reflect the promoter basepair altered in Zp-V3 relative to the Zp start site) and treated with or without anti-IgM. The luciferase activity for each construct is shown and results were normalized to that of each promoter’s untreated condition (set as 1). (C) BJAB cells were transfected with either the wildtype Zp-P luciferase construct or a mutant Zp-P construct in which the A nucleotide located at -141 is switched to the G nucleotide found in the Zp-V3 sequence. Cells were treated with or without anti-IgM 24 hours after transfection. Luciferase activity was measured at 48 hours after transfection and results were normalized to that of each promoter’s untreated condition (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD).
Figure Legend Snippet: Residue -141 in the Zp-V3 promoter is critical for response to BCR crosslinking. (A) The Z promoter sequences in the B95.8 (top) and M81 (bottom) EBV strains are shown. B95.8 is the prototype (Zp-P) while M81 has the 3 polymorphisms at -100, -106, and -141 that define Variant 3 (Zp-V3). There are 4 additional basepair differences between the two Zp sequences, located at -274, -365, -460, and -525 (relative to the transcriptional start site), highlighted in gray. (B) EBV-negative BJAB cells were transfected with wildtype or mutant Zp-V3 luciferase constructs (named to reflect the promoter basepair altered in Zp-V3 relative to the Zp start site) and treated with or without anti-IgM. The luciferase activity for each construct is shown and results were normalized to that of each promoter’s untreated condition (set as 1). (C) BJAB cells were transfected with either the wildtype Zp-P luciferase construct or a mutant Zp-P construct in which the A nucleotide located at -141 is switched to the G nucleotide found in the Zp-V3 sequence. Cells were treated with or without anti-IgM 24 hours after transfection. Luciferase activity was measured at 48 hours after transfection and results were normalized to that of each promoter’s untreated condition (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD).

Techniques Used: Variant Assay, Transfection, Mutagenesis, Luciferase, Construct, Activity Assay, Sequencing

Zp-V3 (but not Zp-P) binds NFATc1. (A) EMSA oligonucleotides were designed to encompass the regions of Zp-V3 and Zp-P from -155 to -127, or from -155 to -120 and radiolabeled with 32 P. The potential NFAT (in Zp-V3, not Zp-P) and AP1 binding sites (in both variants) are indicated. (B) EBV-negative BJAB cells were transfected with the Zp-V3 luciferase vector, and then treated 24 hours later with or without anti-IgM, in the presence or absence of cyclosporine A (1μM) as indicated. Luciferase activity was measured 24 hours after anti-IgM treatment and results were normalized to that of the Zp-V3 untreated condition. (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) Nuclear extracts prepared from BJAB cells, treated with or without anti-IgM for 30 minutes or 6 hours, were incubated with the radiolabeled Zp-P or Zp-V3 (-155 to -127) probes in an EMSA. A protein that binds only to the Zp-V3 probe is indicated by an arrow. (D) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. Cold competitor DNA containing either the Zp-P or Zp-V3 oligonucleotides, or the consensus binding sites for the transcription factors indicated, was added in some conditions. An arrow depicts bands representing NFAT binding. (E) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. In some conditions, antibodies against NFATc1 or C/EBPα were added to the nuclear extract (prior to the addition of the labeled probe) as shown. An arrow depicts bands representing NFAT binding.
Figure Legend Snippet: Zp-V3 (but not Zp-P) binds NFATc1. (A) EMSA oligonucleotides were designed to encompass the regions of Zp-V3 and Zp-P from -155 to -127, or from -155 to -120 and radiolabeled with 32 P. The potential NFAT (in Zp-V3, not Zp-P) and AP1 binding sites (in both variants) are indicated. (B) EBV-negative BJAB cells were transfected with the Zp-V3 luciferase vector, and then treated 24 hours later with or without anti-IgM, in the presence or absence of cyclosporine A (1μM) as indicated. Luciferase activity was measured 24 hours after anti-IgM treatment and results were normalized to that of the Zp-V3 untreated condition. (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) Nuclear extracts prepared from BJAB cells, treated with or without anti-IgM for 30 minutes or 6 hours, were incubated with the radiolabeled Zp-P or Zp-V3 (-155 to -127) probes in an EMSA. A protein that binds only to the Zp-V3 probe is indicated by an arrow. (D) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. Cold competitor DNA containing either the Zp-P or Zp-V3 oligonucleotides, or the consensus binding sites for the transcription factors indicated, was added in some conditions. An arrow depicts bands representing NFAT binding. (E) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. In some conditions, antibodies against NFATc1 or C/EBPα were added to the nuclear extract (prior to the addition of the labeled probe) as shown. An arrow depicts bands representing NFAT binding.

Techniques Used: Binding Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Incubation, Labeling

The longer Zp-V3 probe contains a cooperative NFAT/AP1 binding motif. EBV-negative BJAB nuclear extracts, with or without exposure to 6 hours of anti-IgM, were incubated with radiolabeled probes in EMSA assays. (A) A radiolabeled AP1 consensus probe was incubated with nuclear extract obtained from untreated or anti-IgM treated BJAB cells and EMSA performed. Cold competitor DNA containing consensus binding sites for the AP1 or NFAT transcription factors was added in some conditions. (B) Radiolabeled probes containing the Zp-V3 sequences (either from -155 to -127, or from -155 to -120 as indicated) were incubated with nuclear extract obtained from untreated or anti-IgM treated BJAB cells and EMSA performed. Cold competitor DNA containing consensus binding sites for the AP1 or NFAT transcription factors was added in some conditions. Arrows depict bands representing NFAT binding alone and NFAT plus AP1 binding. (C) A radiolabeled probe containing the Zp-V3 sequence from -155 to -120 was incubated with nuclear extract obtained from untreated or anti-IgM treated BJAB cells and EMSA performed. In some conditions, antibodies against cFos or XBP1 were added to the nuclear extract (prior to the addition of the labeled probe) as shown. Arrows depict bands representing NFAT binding alone, NFAT plus AP1 binding, and the NFAT plus AP1 band that is supershifted by the cFos antibody.
Figure Legend Snippet: The longer Zp-V3 probe contains a cooperative NFAT/AP1 binding motif. EBV-negative BJAB nuclear extracts, with or without exposure to 6 hours of anti-IgM, were incubated with radiolabeled probes in EMSA assays. (A) A radiolabeled AP1 consensus probe was incubated with nuclear extract obtained from untreated or anti-IgM treated BJAB cells and EMSA performed. Cold competitor DNA containing consensus binding sites for the AP1 or NFAT transcription factors was added in some conditions. (B) Radiolabeled probes containing the Zp-V3 sequences (either from -155 to -127, or from -155 to -120 as indicated) were incubated with nuclear extract obtained from untreated or anti-IgM treated BJAB cells and EMSA performed. Cold competitor DNA containing consensus binding sites for the AP1 or NFAT transcription factors was added in some conditions. Arrows depict bands representing NFAT binding alone and NFAT plus AP1 binding. (C) A radiolabeled probe containing the Zp-V3 sequence from -155 to -120 was incubated with nuclear extract obtained from untreated or anti-IgM treated BJAB cells and EMSA performed. In some conditions, antibodies against cFos or XBP1 were added to the nuclear extract (prior to the addition of the labeled probe) as shown. Arrows depict bands representing NFAT binding alone, NFAT plus AP1 binding, and the NFAT plus AP1 band that is supershifted by the cFos antibody.

Techniques Used: Binding Assay, Incubation, Sequencing, Labeling

Zp-V3 is much more responsive to BCR crosslinking than Zp-P. (A) EBV-negative BJAB cells were transfected with a promoterless luciferase construct, or luciferase constructs driven by the Zp-P or Zp-V3 promoters, and luciferase activity was measured after 24 hours. The amount of luciferase activity for each condition (average and the standard deviation, SD) is shown. (B) BJAB cells were transfected with luciferase constructs driven by the Zp-P or Zp-V3 promoters, and then treated with or without anti-human IgM (10 mg/mL) 24 hours later to activate the B cell receptor (BCR). Luciferase activity was measured at 48 hours after transfection and results were normalized to that each promoter’s untreated condition (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) EBV-negative NOKs epithelial cells were transfected with the Zp-P or Zp-V3 luciferase vectors, and a KLF4 expression vector or vector control as indicated, and luciferase activity was measured 24 hours post-transfection. The amount of luciferase activity for each condition (average and SD) is shown and results were normalized to that of each promoter’s untreated condition (set as 1).
Figure Legend Snippet: Zp-V3 is much more responsive to BCR crosslinking than Zp-P. (A) EBV-negative BJAB cells were transfected with a promoterless luciferase construct, or luciferase constructs driven by the Zp-P or Zp-V3 promoters, and luciferase activity was measured after 24 hours. The amount of luciferase activity for each condition (average and the standard deviation, SD) is shown. (B) BJAB cells were transfected with luciferase constructs driven by the Zp-P or Zp-V3 promoters, and then treated with or without anti-human IgM (10 mg/mL) 24 hours later to activate the B cell receptor (BCR). Luciferase activity was measured at 48 hours after transfection and results were normalized to that each promoter’s untreated condition (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) EBV-negative NOKs epithelial cells were transfected with the Zp-P or Zp-V3 luciferase vectors, and a KLF4 expression vector or vector control as indicated, and luciferase activity was measured 24 hours post-transfection. The amount of luciferase activity for each condition (average and SD) is shown and results were normalized to that of each promoter’s untreated condition (set as 1).

Techniques Used: Transfection, Luciferase, Construct, Activity Assay, Standard Deviation, Expressing, Plasmid Preparation

23) Product Images from "Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration"

Article Title: Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration

Journal: Nature Communications

doi: 10.1038/s41467-018-06621-3

SVP[Rapa] treatment enables AAV8 re-administration in nonhuman primates. a Protocol outline. Male naive cynomolgus monkeys were treated i.v. with 2 × 10 12 vg kg −1 of AAV8-Gaa vector and either SVP[Rapa] (3 mg kg −1 , n = 2, SVP[Rapa]#1 and SVP[Rapa]#2) or SVP[empty] control ( n = 1) and then challenged i.v. on day 30 with 2 × 10 12 vg kg − 1 of AAV8-hF.IX vector and either SVP[Rapa] or SVP[empty] control, as described above. b , c Analysis of b anti-AAV8 IgG antibodies and c anti-AAV8 IgM antibody responses measured by ELISA. d Analysis of anti-AAV8 neutralizing antibodies (NAb) measured with a cell-based neutralization assay. e Analysis of anti-AAV8 IgG secreting B cells in splenocytes measured by B ELISpot. Data are shown as individual replicates and the bars represent mean ± s.d. The dotted line indicates the threshold for positivity corresponding to 50 spot forming units (SFU) per million cells. f Plasma hF.IX antigen levels quantified by ELISA at the indicated time points following administration of AAV8-hF.IX vector. g AAV8-hF.IX vector genome copy number (VGCN) per diploid genome in liver. The symbols represent individual liver lobes (left, right, caudate and quadrate) and the bars represent the mean ± s.d. (4 liver lobes per monkey; one-way ANOVA with Tukey post hoc test, ** p
Figure Legend Snippet: SVP[Rapa] treatment enables AAV8 re-administration in nonhuman primates. a Protocol outline. Male naive cynomolgus monkeys were treated i.v. with 2 × 10 12 vg kg −1 of AAV8-Gaa vector and either SVP[Rapa] (3 mg kg −1 , n = 2, SVP[Rapa]#1 and SVP[Rapa]#2) or SVP[empty] control ( n = 1) and then challenged i.v. on day 30 with 2 × 10 12 vg kg − 1 of AAV8-hF.IX vector and either SVP[Rapa] or SVP[empty] control, as described above. b , c Analysis of b anti-AAV8 IgG antibodies and c anti-AAV8 IgM antibody responses measured by ELISA. d Analysis of anti-AAV8 neutralizing antibodies (NAb) measured with a cell-based neutralization assay. e Analysis of anti-AAV8 IgG secreting B cells in splenocytes measured by B ELISpot. Data are shown as individual replicates and the bars represent mean ± s.d. The dotted line indicates the threshold for positivity corresponding to 50 spot forming units (SFU) per million cells. f Plasma hF.IX antigen levels quantified by ELISA at the indicated time points following administration of AAV8-hF.IX vector. g AAV8-hF.IX vector genome copy number (VGCN) per diploid genome in liver. The symbols represent individual liver lobes (left, right, caudate and quadrate) and the bars represent the mean ± s.d. (4 liver lobes per monkey; one-way ANOVA with Tukey post hoc test, ** p

Techniques Used: Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Neutralization, Enzyme-linked Immunospot

Inhibition of anti-AAV8 capsid cellular and humoral responses with SVP[Rapa] co-administration. a CD8 T cell infiltrates in the liver. Livers from animals treated in Fig. 1a were collected after killing on day 53 and evaluated for CD8 mRNA expression by quantitative PCR using the ΔΔC t method relative to housekeeping gene and to average of untreated mice. b – d Male C57BL/6 mice were treated with 4 × 10 12 vg kg − 1 of AAV8-VP1 vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, spleens were collected for B and T cell assays. b Analysis of T cell recall responses after overnight stimulation with an AAV8 peptide pool in splenocytes measured by IFN-γ ELISpot and c anti-AAV8 IgG and IgM secreting B cell responses in splenocytes measured by B ELISpot. d Frequency of B220 + CD19 + B cells in spleens measured by flow cytometry. e – i Male C57BL/6 mice were treated with 4 × 10 12 vg kg − 1 of AAV8-luc vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, animals were sacrificed. e Gating of germinal center (GC) B cells (CD95 + GL7 + ) are shown in representative flow cytometry plot. Cells were gated on B220 + IgD − cells. Shown is a mouse from the SVP[empty] control group. f Frequency of GC B cells (CD95 + GL7 + ) in lymph nodes of treated mice determined by flow cytometry as shown in e ; g frequency of CD25 + FoxP3 + regulatory T cells in lymph nodes; h frequency of CXCR5 + PD1 + Foxp3 + follicular regulatory T (Tfr) cells and i frequency of CXCR5 + PD1 + FoxP3 − follicular helper T (Tfh) cells in lymph nodes. SVP[Rapa] treatment consisted of 200 µg of rapamycin. Data are shown as mean ± s.d. Statistical analyses were performed by one-way ANOVA with Tukey post hoc test in a and by unpaired, two-tail t -test in b – d , f – i ( n = 5 in a – d , n = 20 in f – i . * p
Figure Legend Snippet: Inhibition of anti-AAV8 capsid cellular and humoral responses with SVP[Rapa] co-administration. a CD8 T cell infiltrates in the liver. Livers from animals treated in Fig. 1a were collected after killing on day 53 and evaluated for CD8 mRNA expression by quantitative PCR using the ΔΔC t method relative to housekeeping gene and to average of untreated mice. b – d Male C57BL/6 mice were treated with 4 × 10 12 vg kg − 1 of AAV8-VP1 vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, spleens were collected for B and T cell assays. b Analysis of T cell recall responses after overnight stimulation with an AAV8 peptide pool in splenocytes measured by IFN-γ ELISpot and c anti-AAV8 IgG and IgM secreting B cell responses in splenocytes measured by B ELISpot. d Frequency of B220 + CD19 + B cells in spleens measured by flow cytometry. e – i Male C57BL/6 mice were treated with 4 × 10 12 vg kg − 1 of AAV8-luc vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, animals were sacrificed. e Gating of germinal center (GC) B cells (CD95 + GL7 + ) are shown in representative flow cytometry plot. Cells were gated on B220 + IgD − cells. Shown is a mouse from the SVP[empty] control group. f Frequency of GC B cells (CD95 + GL7 + ) in lymph nodes of treated mice determined by flow cytometry as shown in e ; g frequency of CD25 + FoxP3 + regulatory T cells in lymph nodes; h frequency of CXCR5 + PD1 + Foxp3 + follicular regulatory T (Tfr) cells and i frequency of CXCR5 + PD1 + FoxP3 − follicular helper T (Tfh) cells in lymph nodes. SVP[Rapa] treatment consisted of 200 µg of rapamycin. Data are shown as mean ± s.d. Statistical analyses were performed by one-way ANOVA with Tukey post hoc test in a and by unpaired, two-tail t -test in b – d , f – i ( n = 5 in a – d , n = 20 in f – i . * p

Techniques Used: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Plasmid Preparation, Enzyme-linked Immunospot, Flow Cytometry, Cytometry

24) Product Images from "Serologic features of cohorts with variable genetic risk for systemic lupus erythematosus"

Article Title: Serologic features of cohorts with variable genetic risk for systemic lupus erythematosus

Journal: Molecular Medicine

doi: 10.1186/s10020-018-0019-4

SLE had the highest mean IgG/IgM anti-DNA antibody ratio. The mean ratios of the SIS, AAHC, and MAL cohorts did not significantly differ from each other. The mean ratio for the CHC cohort was significantly lower than all other groups
Figure Legend Snippet: SLE had the highest mean IgG/IgM anti-DNA antibody ratio. The mean ratios of the SIS, AAHC, and MAL cohorts did not significantly differ from each other. The mean ratio for the CHC cohort was significantly lower than all other groups

Techniques Used:

25) Product Images from "A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection"

Article Title: A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007179

Residue -141 in the Zp-V3 promoter is critical for response to BCR crosslinking. (A) The Z promoter sequences in the B95.8 (top) and M81 (bottom) EBV strains are shown. B95.8 is the prototype (Zp-P) while M81 has the 3 polymorphisms at -100, -106, and -141 that define Variant 3 (Zp-V3). There are 4 additional basepair differences between the two Zp sequences, located at -274, -365, -460, and -525 (relative to the transcriptional start site), highlighted in gray. (B) EBV-negative BJAB cells were transfected with wildtype or mutant Zp-V3 luciferase constructs (named to reflect the promoter basepair altered in Zp-V3 relative to the Zp start site) and treated with or without anti-IgM. The luciferase activity for each construct is shown and results were normalized to that of each promoter’s untreated condition (set as 1). (C) BJAB cells were transfected with either the wildtype Zp-P luciferase construct or a mutant Zp-P construct in which the A nucleotide located at -141 is switched to the G nucleotide found in the Zp-V3 sequence. Cells were treated with or without anti-IgM 24 hours after transfection. Luciferase activity was measured at 48 hours after transfection and results were normalized to that of each promoter’s untreated condition (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD).
Figure Legend Snippet: Residue -141 in the Zp-V3 promoter is critical for response to BCR crosslinking. (A) The Z promoter sequences in the B95.8 (top) and M81 (bottom) EBV strains are shown. B95.8 is the prototype (Zp-P) while M81 has the 3 polymorphisms at -100, -106, and -141 that define Variant 3 (Zp-V3). There are 4 additional basepair differences between the two Zp sequences, located at -274, -365, -460, and -525 (relative to the transcriptional start site), highlighted in gray. (B) EBV-negative BJAB cells were transfected with wildtype or mutant Zp-V3 luciferase constructs (named to reflect the promoter basepair altered in Zp-V3 relative to the Zp start site) and treated with or without anti-IgM. The luciferase activity for each construct is shown and results were normalized to that of each promoter’s untreated condition (set as 1). (C) BJAB cells were transfected with either the wildtype Zp-P luciferase construct or a mutant Zp-P construct in which the A nucleotide located at -141 is switched to the G nucleotide found in the Zp-V3 sequence. Cells were treated with or without anti-IgM 24 hours after transfection. Luciferase activity was measured at 48 hours after transfection and results were normalized to that of each promoter’s untreated condition (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD).

Techniques Used: Variant Assay, Transfection, Mutagenesis, Luciferase, Construct, Activity Assay, Sequencing

Zp-V3 (but not Zp-P) binds NFATc1. (A) EMSA oligonucleotides were designed to encompass the regions of Zp-V3 and Zp-P from -155 to -127, or from -155 to -120 and radiolabeled with 32 P. The potential NFAT (in Zp-V3, not Zp-P) and AP1 binding sites (in both variants) are indicated. (B) EBV-negative BJAB cells were transfected with the Zp-V3 luciferase vector, and then treated 24 hours later with or without anti-IgM, in the presence or absence of cyclosporine A (1μM) as indicated. Luciferase activity was measured 24 hours after anti-IgM treatment and results were normalized to that of the Zp-V3 untreated condition. (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) Nuclear extracts prepared from BJAB cells, treated with or without anti-IgM for 30 minutes or 6 hours, were incubated with the radiolabeled Zp-P or Zp-V3 (-155 to -127) probes in an EMSA. A protein that binds only to the Zp-V3 probe is indicated by an arrow. (D) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. Cold competitor DNA containing either the Zp-P or Zp-V3 oligonucleotides, or the consensus binding sites for the transcription factors indicated, was added in some conditions. An arrow depicts bands representing NFAT binding. (E) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. In some conditions, antibodies against NFATc1 or C/EBPα were added to the nuclear extract (prior to the addition of the labeled probe) as shown. An arrow depicts bands representing NFAT binding.
Figure Legend Snippet: Zp-V3 (but not Zp-P) binds NFATc1. (A) EMSA oligonucleotides were designed to encompass the regions of Zp-V3 and Zp-P from -155 to -127, or from -155 to -120 and radiolabeled with 32 P. The potential NFAT (in Zp-V3, not Zp-P) and AP1 binding sites (in both variants) are indicated. (B) EBV-negative BJAB cells were transfected with the Zp-V3 luciferase vector, and then treated 24 hours later with or without anti-IgM, in the presence or absence of cyclosporine A (1μM) as indicated. Luciferase activity was measured 24 hours after anti-IgM treatment and results were normalized to that of the Zp-V3 untreated condition. (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) Nuclear extracts prepared from BJAB cells, treated with or without anti-IgM for 30 minutes or 6 hours, were incubated with the radiolabeled Zp-P or Zp-V3 (-155 to -127) probes in an EMSA. A protein that binds only to the Zp-V3 probe is indicated by an arrow. (D) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. Cold competitor DNA containing either the Zp-P or Zp-V3 oligonucleotides, or the consensus binding sites for the transcription factors indicated, was added in some conditions. An arrow depicts bands representing NFAT binding. (E) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. In some conditions, antibodies against NFATc1 or C/EBPα were added to the nuclear extract (prior to the addition of the labeled probe) as shown. An arrow depicts bands representing NFAT binding.

Techniques Used: Binding Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Incubation, Labeling

The longer Zp-V3 probe contains a cooperative NFAT/AP1 binding motif. EBV-negative BJAB nuclear extracts, with or without exposure to 6 hours of anti-IgM, were incubated with radiolabeled probes in EMSA assays. (A) A radiolabeled AP1 consensus probe was incubated with nuclear extract obtained from untreated or anti-IgM treated BJAB cells and EMSA performed. Cold competitor DNA containing consensus binding sites for the AP1 or NFAT transcription factors was added in some conditions. (B) Radiolabeled probes containing the Zp-V3 sequences (either from -155 to -127, or from -155 to -120 as indicated) were incubated with nuclear extract obtained from untreated or anti-IgM treated BJAB cells and EMSA performed. Cold competitor DNA containing consensus binding sites for the AP1 or NFAT transcription factors was added in some conditions. Arrows depict bands representing NFAT binding alone and NFAT plus AP1 binding. (C) A radiolabeled probe containing the Zp-V3 sequence from -155 to -120 was incubated with nuclear extract obtained from untreated or anti-IgM treated BJAB cells and EMSA performed. In some conditions, antibodies against cFos or XBP1 were added to the nuclear extract (prior to the addition of the labeled probe) as shown. Arrows depict bands representing NFAT binding alone, NFAT plus AP1 binding, and the NFAT plus AP1 band that is supershifted by the cFos antibody.
Figure Legend Snippet: The longer Zp-V3 probe contains a cooperative NFAT/AP1 binding motif. EBV-negative BJAB nuclear extracts, with or without exposure to 6 hours of anti-IgM, were incubated with radiolabeled probes in EMSA assays. (A) A radiolabeled AP1 consensus probe was incubated with nuclear extract obtained from untreated or anti-IgM treated BJAB cells and EMSA performed. Cold competitor DNA containing consensus binding sites for the AP1 or NFAT transcription factors was added in some conditions. (B) Radiolabeled probes containing the Zp-V3 sequences (either from -155 to -127, or from -155 to -120 as indicated) were incubated with nuclear extract obtained from untreated or anti-IgM treated BJAB cells and EMSA performed. Cold competitor DNA containing consensus binding sites for the AP1 or NFAT transcription factors was added in some conditions. Arrows depict bands representing NFAT binding alone and NFAT plus AP1 binding. (C) A radiolabeled probe containing the Zp-V3 sequence from -155 to -120 was incubated with nuclear extract obtained from untreated or anti-IgM treated BJAB cells and EMSA performed. In some conditions, antibodies against cFos or XBP1 were added to the nuclear extract (prior to the addition of the labeled probe) as shown. Arrows depict bands representing NFAT binding alone, NFAT plus AP1 binding, and the NFAT plus AP1 band that is supershifted by the cFos antibody.

Techniques Used: Binding Assay, Incubation, Sequencing, Labeling

Zp-V3 is much more responsive to BCR crosslinking than Zp-P. (A) EBV-negative BJAB cells were transfected with a promoterless luciferase construct, or luciferase constructs driven by the Zp-P or Zp-V3 promoters, and luciferase activity was measured after 24 hours. The amount of luciferase activity for each condition (average and the standard deviation, SD) is shown. (B) BJAB cells were transfected with luciferase constructs driven by the Zp-P or Zp-V3 promoters, and then treated with or without anti-human IgM (10 mg/mL) 24 hours later to activate the B cell receptor (BCR). Luciferase activity was measured at 48 hours after transfection and results were normalized to that each promoter’s untreated condition (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) EBV-negative NOKs epithelial cells were transfected with the Zp-P or Zp-V3 luciferase vectors, and a KLF4 expression vector or vector control as indicated, and luciferase activity was measured 24 hours post-transfection. The amount of luciferase activity for each condition (average and SD) is shown and results were normalized to that of each promoter’s untreated condition (set as 1).
Figure Legend Snippet: Zp-V3 is much more responsive to BCR crosslinking than Zp-P. (A) EBV-negative BJAB cells were transfected with a promoterless luciferase construct, or luciferase constructs driven by the Zp-P or Zp-V3 promoters, and luciferase activity was measured after 24 hours. The amount of luciferase activity for each condition (average and the standard deviation, SD) is shown. (B) BJAB cells were transfected with luciferase constructs driven by the Zp-P or Zp-V3 promoters, and then treated with or without anti-human IgM (10 mg/mL) 24 hours later to activate the B cell receptor (BCR). Luciferase activity was measured at 48 hours after transfection and results were normalized to that each promoter’s untreated condition (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) EBV-negative NOKs epithelial cells were transfected with the Zp-P or Zp-V3 luciferase vectors, and a KLF4 expression vector or vector control as indicated, and luciferase activity was measured 24 hours post-transfection. The amount of luciferase activity for each condition (average and SD) is shown and results were normalized to that of each promoter’s untreated condition (set as 1).

Techniques Used: Transfection, Luciferase, Construct, Activity Assay, Standard Deviation, Expressing, Plasmid Preparation

26) Product Images from "Blockade of B-cell-activating factor suppresses lupus-like syndrome in autoimmune BXSB mice"

Article Title: Blockade of B-cell-activating factor suppresses lupus-like syndrome in autoimmune BXSB mice

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2009.00817.x

Characterization of BAFF-R-IgG4mut fusion protein. (A) SDS-PAGE analysis. A and B both are loaded with purified BAFF-R-IgG4mut. (B) Western blot analysis with anti-BAFF-R mAb. 1, negative control; 2, BAFF-R-IgG4mut fusion protein; M, molecular weight marker. (C) In vitro bioactivity assay. Purified mouse splenic B cells were stimulated with 5 μg/ml (left side) or 10 μg/ml (right side) of anti-IgM in the presence of 2 ng/ml of BAFF and indicated doses of BAFF-R-IgG4mut fusion protein. B-cell proliferation after 3 days was measured by 3 H-thymidine incorporation in triplicates. B-cell thymidine incorporation by anti-IgM stimulation alone was labelled as a flat line in each side. BAFF-R-IgG4mut alone did not stimulate B-cell proliferation (data not shown).
Figure Legend Snippet: Characterization of BAFF-R-IgG4mut fusion protein. (A) SDS-PAGE analysis. A and B both are loaded with purified BAFF-R-IgG4mut. (B) Western blot analysis with anti-BAFF-R mAb. 1, negative control; 2, BAFF-R-IgG4mut fusion protein; M, molecular weight marker. (C) In vitro bioactivity assay. Purified mouse splenic B cells were stimulated with 5 μg/ml (left side) or 10 μg/ml (right side) of anti-IgM in the presence of 2 ng/ml of BAFF and indicated doses of BAFF-R-IgG4mut fusion protein. B-cell proliferation after 3 days was measured by 3 H-thymidine incorporation in triplicates. B-cell thymidine incorporation by anti-IgM stimulation alone was labelled as a flat line in each side. BAFF-R-IgG4mut alone did not stimulate B-cell proliferation (data not shown).

Techniques Used: SDS Page, Purification, Western Blot, Negative Control, Molecular Weight, Marker, In Vitro

27) Product Images from "Malaria-associated atypical memory B cells exhibit markedly reduced B cell receptor signaling and effector function"

Article Title: Malaria-associated atypical memory B cells exhibit markedly reduced B cell receptor signaling and effector function

Journal: eLife

doi: 10.7554/eLife.07218

Atypical MBCs do not actively secrete Abs or differentiate into ASCs. ( A ) Total PBMCs and magnetically sorted naïve B cells (CD19 + CD21 + CD27 − ), classical (CD19 + CD21 + CD27 + ) and atypical MBCs (CD19 + CD21 − CD27 − ) were cultured for 48 hr without stimulation, then ASCs were quantified by IgM/IgA/IgG ELISPOT (n = 14 subjects). ( B ) Magnetic sorting strategy for experiments described in D and E . B cells without plasma cells/plasmablasts were negatively selected with Abs to CD2, CD3, CD14, CD16, CD36, CD43, CD56, CD66b, and glycophorin A (left panel, blue population), while total B cells were negatively selected with Abs to CD2, CD3, CD14, CD16, CD56, and glycophorin A (left panel, red population). Red population contains plasma cells/plasmablasts (CD19 + CD20 − CD21 − ) (middle panel, aqua population) whereas blue population does not (right panel). ( C ) Representative histogram showing specificity of CD43 for plasma cells/plasmablasts. ( D ) Total B cells (CD43 +/− fraction) and B cells without plasma cells/plasmablasts (CD43 − fraction) were isolated from Malian adults and cultured for 48 hr without stimulation, then ASCs were quantified by IgM/IgA/IgG ELISPOT (n = 6 subjects). ( E ) Total B cells and B cells without plasma cells/plasmablasts were isolated from children during acute malaria and cultured for 48 hr without stimulation, then ASCs were quantified by IgM/IgA/IgG ELISPOT (n = 10 subjects). ( F ) Magnetically sorted B cell subpopulations were cultured for 6 days with CpG, IL-10, SAC and PWM and ASCs were quantified by IgM/IgA/IgG ELISPOT (n = 9 subjects). Horizontal bars and whiskers represent means and SD, respectively. p values determined by ANOVA with Tukey's multiple comparisons test ( A and F ) or paired ttest ( D and E ). DOI: http://dx.doi.org/10.7554/eLife.07218.012
Figure Legend Snippet: Atypical MBCs do not actively secrete Abs or differentiate into ASCs. ( A ) Total PBMCs and magnetically sorted naïve B cells (CD19 + CD21 + CD27 − ), classical (CD19 + CD21 + CD27 + ) and atypical MBCs (CD19 + CD21 − CD27 − ) were cultured for 48 hr without stimulation, then ASCs were quantified by IgM/IgA/IgG ELISPOT (n = 14 subjects). ( B ) Magnetic sorting strategy for experiments described in D and E . B cells without plasma cells/plasmablasts were negatively selected with Abs to CD2, CD3, CD14, CD16, CD36, CD43, CD56, CD66b, and glycophorin A (left panel, blue population), while total B cells were negatively selected with Abs to CD2, CD3, CD14, CD16, CD56, and glycophorin A (left panel, red population). Red population contains plasma cells/plasmablasts (CD19 + CD20 − CD21 − ) (middle panel, aqua population) whereas blue population does not (right panel). ( C ) Representative histogram showing specificity of CD43 for plasma cells/plasmablasts. ( D ) Total B cells (CD43 +/− fraction) and B cells without plasma cells/plasmablasts (CD43 − fraction) were isolated from Malian adults and cultured for 48 hr without stimulation, then ASCs were quantified by IgM/IgA/IgG ELISPOT (n = 6 subjects). ( E ) Total B cells and B cells without plasma cells/plasmablasts were isolated from children during acute malaria and cultured for 48 hr without stimulation, then ASCs were quantified by IgM/IgA/IgG ELISPOT (n = 10 subjects). ( F ) Magnetically sorted B cell subpopulations were cultured for 6 days with CpG, IL-10, SAC and PWM and ASCs were quantified by IgM/IgA/IgG ELISPOT (n = 9 subjects). Horizontal bars and whiskers represent means and SD, respectively. p values determined by ANOVA with Tukey's multiple comparisons test ( A and F ) or paired ttest ( D and E ). DOI: http://dx.doi.org/10.7554/eLife.07218.012

Techniques Used: Cell Culture, Enzyme-linked Immunospot, Isolation

FcRL5 expression on atypical MBCs is associated with markedly reduced BCR signaling. ( A ) Following 5 min of BCR cross-linking with anti-IgM and anti-IgG Abs, PBMCs were incubated with Abs specific for phosphorylated PLCγ2 (left) and Syk (right). Classical MBCs, and FcRL5 − and FcRL5 + atypical MBCs were analyzed by flow cytometry. Shown are ratios of phosphorylation induced by BCR cross-linking over tonic signal fluorescence in RPMI (n = 7 subjects). ( B ) MFI of FcγR2b expression by flow cytometry on classical MBCs, and FcRL5 − and FcRL5 + atypical MBCs (n = 10 subjects). Representative MFI histogram shown on left. Horizontal bars and whiskers represent means and SD, respectively. p values determined by ANOVA with Tukey's multiple comparisons test ( A and B ). DOI: http://dx.doi.org/10.7554/eLife.07218.011
Figure Legend Snippet: FcRL5 expression on atypical MBCs is associated with markedly reduced BCR signaling. ( A ) Following 5 min of BCR cross-linking with anti-IgM and anti-IgG Abs, PBMCs were incubated with Abs specific for phosphorylated PLCγ2 (left) and Syk (right). Classical MBCs, and FcRL5 − and FcRL5 + atypical MBCs were analyzed by flow cytometry. Shown are ratios of phosphorylation induced by BCR cross-linking over tonic signal fluorescence in RPMI (n = 7 subjects). ( B ) MFI of FcγR2b expression by flow cytometry on classical MBCs, and FcRL5 − and FcRL5 + atypical MBCs (n = 10 subjects). Representative MFI histogram shown on left. Horizontal bars and whiskers represent means and SD, respectively. p values determined by ANOVA with Tukey's multiple comparisons test ( A and B ). DOI: http://dx.doi.org/10.7554/eLife.07218.011

Techniques Used: Expressing, Incubation, Flow Cytometry, Cytometry, Fluorescence

28) Product Images from "Cell-intrinsic determinants of ibrutinib-induced apoptosis in Chronic Lymphocytic Leukemia"

Article Title: Cell-intrinsic determinants of ibrutinib-induced apoptosis in Chronic Lymphocytic Leukemia

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-15-2921

A–B: Results of ex vivo B-cell receptor signaling studies in CLL Purified CLL cells were cultured in serum-free medium for 2 hours and left untreated, or pre-treated with ibrutinib at 0 μM (DMSO only) or 0.25 μM, 0.5 μM or 1 μM for 60′ and subsequently treated with anti-IgM at 10μg/ml for 15′. Cells were pelleted, lysates made, protein fractionated and prepared for immunoblotting with antibodies to p1217-PLCy2; PLCy2; p223-BTK; BTK; p473-AKT; AKT; p202/204-ERK and ERK. Figure 5; Panel A (del17p/TP53 mutated CLL) and Figure 5; Panel B (non-del17p/TP53mutated CLL). The results for individual blots and epitopes cannot be directly quantitatively compared across different patients as exposure times for various blots are different. The IGVH mutation status for each CLL case is indicated (UM: unmutated; M: mutated). The ex vivo IC50 values to ibrutinib are indicated in brackets.
Figure Legend Snippet: A–B: Results of ex vivo B-cell receptor signaling studies in CLL Purified CLL cells were cultured in serum-free medium for 2 hours and left untreated, or pre-treated with ibrutinib at 0 μM (DMSO only) or 0.25 μM, 0.5 μM or 1 μM for 60′ and subsequently treated with anti-IgM at 10μg/ml for 15′. Cells were pelleted, lysates made, protein fractionated and prepared for immunoblotting with antibodies to p1217-PLCy2; PLCy2; p223-BTK; BTK; p473-AKT; AKT; p202/204-ERK and ERK. Figure 5; Panel A (del17p/TP53 mutated CLL) and Figure 5; Panel B (non-del17p/TP53mutated CLL). The results for individual blots and epitopes cannot be directly quantitatively compared across different patients as exposure times for various blots are different. The IGVH mutation status for each CLL case is indicated (UM: unmutated; M: mutated). The ex vivo IC50 values to ibrutinib are indicated in brackets.

Techniques Used: Ex Vivo, Purification, Cell Culture, Mutagenesis

29) Product Images from "B cell-intrinsic TLR signals amplify but are not required for humoral immunity"

Article Title: B cell-intrinsic TLR signals amplify but are not required for humoral immunity

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20071250

LPS promotes a B cell–intrinsic MyD88-dependent increase in specific antibody titers. (A) Schema for cell transfer and immunization. (B and C) MyD88 WT, Het, or KO B cells were transferred into μMT mice, and recipients were immunized as in A. 8 mo after transfer, 5 μg LPS was injected. (B) NP-specific IgM and (C) IgG before and 1 wk after LPS injection. *, P
Figure Legend Snippet: LPS promotes a B cell–intrinsic MyD88-dependent increase in specific antibody titers. (A) Schema for cell transfer and immunization. (B and C) MyD88 WT, Het, or KO B cells were transferred into μMT mice, and recipients were immunized as in A. 8 mo after transfer, 5 μg LPS was injected. (B) NP-specific IgM and (C) IgG before and 1 wk after LPS injection. *, P

Techniques Used: Mouse Assay, Injection

The addition of LPS promotes B cell–intrinsic MyD88-dependent IgM responses. WT or MyD88 KO B cells were transferred into μMT mice, and recipients were immunized with NP-CGG in alum with or without LPS (5 μg/mouse). (A–D) Total and NP-specific IgM and IgG 1 wk after immunization. (E–H) Total and NP-specific IgG, IgG1, and IgG2a 2 wk after immunization. Data are a summary of four independent experiments with a total of 14 mice per group. *, P
Figure Legend Snippet: The addition of LPS promotes B cell–intrinsic MyD88-dependent IgM responses. WT or MyD88 KO B cells were transferred into μMT mice, and recipients were immunized with NP-CGG in alum with or without LPS (5 μg/mouse). (A–D) Total and NP-specific IgM and IgG 1 wk after immunization. (E–H) Total and NP-specific IgG, IgG1, and IgG2a 2 wk after immunization. Data are a summary of four independent experiments with a total of 14 mice per group. *, P

Techniques Used: Mouse Assay

B cell–intrinsic MyD88 signals are not required for primary or memory responses. Splenic B cells from MyD88 WT, Het, and KO littermates were transferred into μMT mice. Mice were immunized with NP-CGG in alum, and IgM and IgG levels were determined at 1 and 2 wk after immunization, respectively. (A and B) Preimmunization IgM and IgG, (C and D) NP-specific IgM and IgG before and after immunization, and (E and F) NP-specific IgG2a and IgG1 2 wk after immunization. (G and H) 4 mo after secondary immunization, mice were challenged with NP-CGG in PBS, and antibody levels for NP-specific IgM and IgG were determined. Data from one of three independent experiments are shown. Total number of animals: WT, 12; Het, 8; KO,16.
Figure Legend Snippet: B cell–intrinsic MyD88 signals are not required for primary or memory responses. Splenic B cells from MyD88 WT, Het, and KO littermates were transferred into μMT mice. Mice were immunized with NP-CGG in alum, and IgM and IgG levels were determined at 1 and 2 wk after immunization, respectively. (A and B) Preimmunization IgM and IgG, (C and D) NP-specific IgM and IgG before and after immunization, and (E and F) NP-specific IgG2a and IgG1 2 wk after immunization. (G and H) 4 mo after secondary immunization, mice were challenged with NP-CGG in PBS, and antibody levels for NP-specific IgM and IgG were determined. Data from one of three independent experiments are shown. Total number of animals: WT, 12; Het, 8; KO,16.

Techniques Used: Mouse Assay

30) Product Images from "Isolation of immune-regulatory Tetragenococcus halophilus from miso"

Article Title: Isolation of immune-regulatory Tetragenococcus halophilus from miso

Journal: PLoS ONE

doi: 10.1371/journal.pone.0208821

The effect of T . halophilus on the serum IgA, IgM, and IgG in mice. Diet containing 1% T . halophilus were fed to C57BL/6 mice for 2 weeks. Then, the serum samples were obtained, and the serum IgA, IgM, and IgG levels were analyzed by ELISA. Mice fed without T . halophilus were used as control. Bars indicate mean ± S.D. (n = 3 mice). The results of two experiments are shown. *p
Figure Legend Snippet: The effect of T . halophilus on the serum IgA, IgM, and IgG in mice. Diet containing 1% T . halophilus were fed to C57BL/6 mice for 2 weeks. Then, the serum samples were obtained, and the serum IgA, IgM, and IgG levels were analyzed by ELISA. Mice fed without T . halophilus were used as control. Bars indicate mean ± S.D. (n = 3 mice). The results of two experiments are shown. *p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

31) Product Images from "Blockade of B-cell-activating factor suppresses lupus-like syndrome in autoimmune BXSB mice"

Article Title: Blockade of B-cell-activating factor suppresses lupus-like syndrome in autoimmune BXSB mice

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2009.00817.x

Characterization of BAFF-R-IgG4mut fusion protein. (A) SDS-PAGE analysis. A and B both are loaded with purified BAFF-R-IgG4mut. (B) Western blot analysis with anti-BAFF-R mAb. 1, negative control; 2, BAFF-R-IgG4mut fusion protein; M, molecular weight marker. (C) In vitro bioactivity assay. Purified mouse splenic B cells were stimulated with 5 μg/ml (left side) or 10 μg/ml (right side) of anti-IgM in the presence of 2 ng/ml of BAFF and indicated doses of BAFF-R-IgG4mut fusion protein. B-cell proliferation after 3 days was measured by 3 H-thymidine incorporation in triplicates. B-cell thymidine incorporation by anti-IgM stimulation alone was labelled as a flat line in each side. BAFF-R-IgG4mut alone did not stimulate B-cell proliferation (data not shown).
Figure Legend Snippet: Characterization of BAFF-R-IgG4mut fusion protein. (A) SDS-PAGE analysis. A and B both are loaded with purified BAFF-R-IgG4mut. (B) Western blot analysis with anti-BAFF-R mAb. 1, negative control; 2, BAFF-R-IgG4mut fusion protein; M, molecular weight marker. (C) In vitro bioactivity assay. Purified mouse splenic B cells were stimulated with 5 μg/ml (left side) or 10 μg/ml (right side) of anti-IgM in the presence of 2 ng/ml of BAFF and indicated doses of BAFF-R-IgG4mut fusion protein. B-cell proliferation after 3 days was measured by 3 H-thymidine incorporation in triplicates. B-cell thymidine incorporation by anti-IgM stimulation alone was labelled as a flat line in each side. BAFF-R-IgG4mut alone did not stimulate B-cell proliferation (data not shown).

Techniques Used: SDS Page, Purification, Western Blot, Negative Control, Molecular Weight, Marker, In Vitro

32) Product Images from "TACI Isoforms Regulate Ligand Binding and Receptor Function"

Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02125

TACI isoforms expression in activated B cells. TACI isoform expression in non-permeabilized (A) and permeabilized (B) peripheral blood B cells from normal donors, cultured for 4 days in the presence or absence of ODN, CD40L/IL21, or anti-IgM. Data shows the mean ± SEM of 4 independent experiments. (C,D) TACI isoform expression was analyzed in B cell subpopulations isolated from freshly-isolated tonsils ( n = 4): Naïve (IgD+CD27-), Transitional (Tr) (IgM+CD38+), Marginal Zone (MZ) (IgD+CD27+), Switched Memory (SwMe) (IgD-CD27+), and Plasmablasts (PB) (IgD-CD27 hi CD38 hi ). Non permeabilized (C) and permeabilized cells (D) were examined. Data shows the mean ± SEM. * p
Figure Legend Snippet: TACI isoforms expression in activated B cells. TACI isoform expression in non-permeabilized (A) and permeabilized (B) peripheral blood B cells from normal donors, cultured for 4 days in the presence or absence of ODN, CD40L/IL21, or anti-IgM. Data shows the mean ± SEM of 4 independent experiments. (C,D) TACI isoform expression was analyzed in B cell subpopulations isolated from freshly-isolated tonsils ( n = 4): Naïve (IgD+CD27-), Transitional (Tr) (IgM+CD38+), Marginal Zone (MZ) (IgD+CD27+), Switched Memory (SwMe) (IgD-CD27+), and Plasmablasts (PB) (IgD-CD27 hi CD38 hi ). Non permeabilized (C) and permeabilized cells (D) were examined. Data shows the mean ± SEM. * p

Techniques Used: Expressing, Cell Culture, Isolation

33) Product Images from "Circulating CD23+ B Cell Subset Correlates with the Development of Resistance to Schistosoma mansoni Reinfection in Occupationally Exposed Adults Who Have Undergone Multiple Treatments"

Article Title: Circulating CD23+ B Cell Subset Correlates with the Development of Resistance to Schistosoma mansoni Reinfection in Occupationally Exposed Adults Who Have Undergone Multiple Treatments

Journal: The Journal of infectious diseases

doi: 10.1086/595792

CD23 + B cells in human schistosomiasis. a, Flow cytometry analysis of whole-blood cells showing the R1 lymphocyte gate used for all subsequent analyses. Cells stained with anti-CD19 (b), anti-CD23 (c), anti-CD27 (d), anti-IgE (e), and anti-IgM (f) were
Figure Legend Snippet: CD23 + B cells in human schistosomiasis. a, Flow cytometry analysis of whole-blood cells showing the R1 lymphocyte gate used for all subsequent analyses. Cells stained with anti-CD19 (b), anti-CD23 (c), anti-CD27 (d), anti-IgE (e), and anti-IgM (f) were

Techniques Used: Flow Cytometry, Cytometry, Staining

34) Product Images from "Dysregulated TGF-β Production Underlies the Age-Related Vulnerability to Chikungunya Virus"

Article Title: Dysregulated TGF-β Production Underlies the Age-Related Vulnerability to Chikungunya Virus

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005891

Age-related impairment of adaptive immune response to CHIKV. (A) Popliteal LNs collected and quantified from either naïve or CHIKV-infected A and O mice at day 3, 7, or 9 post-infection. The LN draining from the CHIKV-inoculated foot is indicated as dLN and from the non-inoculated foot as ndLN. Table under graph indicates the average fold-increase from naïve for each age in either the dLN or ndLN ( n = 6–8 per group). Horizontal lines indicate the median. Statistical significance determined by student’s t -test. (B-C) Lymphocytes from popliteal LNs on d7 post-infection were stimulated with CHIKV peptides in the presence of protein transport inhibitor. Total number of IFNγ + CD4 T cells (B) and frequency (C) for each age. Data are mean ± SEM ( n = 10 per group). Statistical significance determined by unpaired Student’s t -test. (D) CHIKV-specific IgM and (E) IgG2c in serum determined by ELISA at the indicated day post-infection. Data are mean ( n = 4–24 per group). Statistical significance was determined by two-way ANOVA with Bonferroni post-test. (F) Plaque reduction neutralizing test on serum from days 9 and 60 post-infection. Data are mean + SEM ( n = 12 per group). Statistical significance was determined by two-way ANOVA with Bonferroni post-test. (G) Serum samples collected from patients experiencing acute CHIKV-disease were evaluated by plaque reduction neutralizing test. Data are mean + SEM ( n = 24 young and 15 aged). Statistical significance was evaluated by unpaired student’s t -test. In all panels black indicates A, red indicates O; * P
Figure Legend Snippet: Age-related impairment of adaptive immune response to CHIKV. (A) Popliteal LNs collected and quantified from either naïve or CHIKV-infected A and O mice at day 3, 7, or 9 post-infection. The LN draining from the CHIKV-inoculated foot is indicated as dLN and from the non-inoculated foot as ndLN. Table under graph indicates the average fold-increase from naïve for each age in either the dLN or ndLN ( n = 6–8 per group). Horizontal lines indicate the median. Statistical significance determined by student’s t -test. (B-C) Lymphocytes from popliteal LNs on d7 post-infection were stimulated with CHIKV peptides in the presence of protein transport inhibitor. Total number of IFNγ + CD4 T cells (B) and frequency (C) for each age. Data are mean ± SEM ( n = 10 per group). Statistical significance determined by unpaired Student’s t -test. (D) CHIKV-specific IgM and (E) IgG2c in serum determined by ELISA at the indicated day post-infection. Data are mean ( n = 4–24 per group). Statistical significance was determined by two-way ANOVA with Bonferroni post-test. (F) Plaque reduction neutralizing test on serum from days 9 and 60 post-infection. Data are mean + SEM ( n = 12 per group). Statistical significance was determined by two-way ANOVA with Bonferroni post-test. (G) Serum samples collected from patients experiencing acute CHIKV-disease were evaluated by plaque reduction neutralizing test. Data are mean + SEM ( n = 24 young and 15 aged). Statistical significance was evaluated by unpaired student’s t -test. In all panels black indicates A, red indicates O; * P

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

35) Product Images from "A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection"

Article Title: A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007179

Zp-V3 (but not Zp-P) binds NFATc1. (A) EMSA oligonucleotides were designed to encompass the regions of Zp-V3 and Zp-P from -155 to -127, or from -155 to -120 and radiolabeled with 32 P. The potential NFAT (in Zp-V3, not Zp-P) and AP1 binding sites (in both variants) are indicated. (B) EBV-negative BJAB cells were transfected with the Zp-V3 luciferase vector, and then treated 24 hours later with or without anti-IgM, in the presence or absence of cyclosporine A (1μM) as indicated. Luciferase activity was measured 24 hours after anti-IgM treatment and results were normalized to that of the Zp-V3 untreated condition. (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) Nuclear extracts prepared from BJAB cells, treated with or without anti-IgM for 30 minutes or 6 hours, were incubated with the radiolabeled Zp-P or Zp-V3 (-155 to -127) probes in an EMSA. A protein that binds only to the Zp-V3 probe is indicated by an arrow. (D) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. Cold competitor DNA containing either the Zp-P or Zp-V3 oligonucleotides, or the consensus binding sites for the transcription factors indicated, was added in some conditions. An arrow depicts bands representing NFAT binding. (E) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. In some conditions, antibodies against NFATc1 or C/EBPα were added to the nuclear extract (prior to the addition of the labeled probe) as shown. An arrow depicts bands representing NFAT binding.
Figure Legend Snippet: Zp-V3 (but not Zp-P) binds NFATc1. (A) EMSA oligonucleotides were designed to encompass the regions of Zp-V3 and Zp-P from -155 to -127, or from -155 to -120 and radiolabeled with 32 P. The potential NFAT (in Zp-V3, not Zp-P) and AP1 binding sites (in both variants) are indicated. (B) EBV-negative BJAB cells were transfected with the Zp-V3 luciferase vector, and then treated 24 hours later with or without anti-IgM, in the presence or absence of cyclosporine A (1μM) as indicated. Luciferase activity was measured 24 hours after anti-IgM treatment and results were normalized to that of the Zp-V3 untreated condition. (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) Nuclear extracts prepared from BJAB cells, treated with or without anti-IgM for 30 minutes or 6 hours, were incubated with the radiolabeled Zp-P or Zp-V3 (-155 to -127) probes in an EMSA. A protein that binds only to the Zp-V3 probe is indicated by an arrow. (D) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. Cold competitor DNA containing either the Zp-P or Zp-V3 oligonucleotides, or the consensus binding sites for the transcription factors indicated, was added in some conditions. An arrow depicts bands representing NFAT binding. (E) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. In some conditions, antibodies against NFATc1 or C/EBPα were added to the nuclear extract (prior to the addition of the labeled probe) as shown. An arrow depicts bands representing NFAT binding.

Techniques Used: Binding Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Incubation, Labeling

36) Product Images from "IL-17 augments B cell activation in ocular surface autoimmunity"

Article Title: IL-17 augments B cell activation in ocular surface autoimmunity

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

doi: 10.4049/jimmunol.1502641

B cell proliferation induced by IgM/CD40 stimulation is enhanced by co-stimulation with IL-17 only in autoimmune B cells (A) anti-IgM/anti-CD40–stimulated B cells were cultured at the bottom of the transwell, and anti-CD3-stimulated T effector cells were cultured in the insert (top) in the presence or absence of anti-IL-17A antibody. Flow cytometric analysis showing Ki67 staining of purified B cells from dry eye (DED) mice. (B) Naive or DED B cells were stimulated with anti-IgM/anti-CD40 and with or without recombinant IL-17A. Flow cytometric analysis showing Ki67 staining of purified B cells from DED and naïve mice after 48 hrs stimulation. B cell proliferation is shown as fold change (B cell+ anti-IgM and anti-CD40 =1). All results are representative of three independent experiments. (mean ± SEM; n=4–6 mice/group; triplicate cultures). Student’s t test **, P
Figure Legend Snippet: B cell proliferation induced by IgM/CD40 stimulation is enhanced by co-stimulation with IL-17 only in autoimmune B cells (A) anti-IgM/anti-CD40–stimulated B cells were cultured at the bottom of the transwell, and anti-CD3-stimulated T effector cells were cultured in the insert (top) in the presence or absence of anti-IL-17A antibody. Flow cytometric analysis showing Ki67 staining of purified B cells from dry eye (DED) mice. (B) Naive or DED B cells were stimulated with anti-IgM/anti-CD40 and with or without recombinant IL-17A. Flow cytometric analysis showing Ki67 staining of purified B cells from DED and naïve mice after 48 hrs stimulation. B cell proliferation is shown as fold change (B cell+ anti-IgM and anti-CD40 =1). All results are representative of three independent experiments. (mean ± SEM; n=4–6 mice/group; triplicate cultures). Student’s t test **, P

Techniques Used: Cell Culture, Flow Cytometry, Staining, Purification, Mouse Assay, Recombinant

Activated B cells show enhanced IL-17 receptor expression (A) Immunohistochemistry was performed on submandibular lymph nodes of naïve and dry eye (DED) mice (21 days after DED induction) to visualize Ki67 (proliferating cells, green), IgM (B cells, red), and IL-17RA (receptor expression, blue) expression. Data are representative of 4–6 mice examined. Images are 10x magnification and 20x magnification for magnified view of areas in white box. (B) Flow cytomteric analysis showing IL-17RA+ CD19+ cells after stimulating purified B cells with medium alone or in the presence or absence of anti-IgM, anti-CD40, and IL-17A for 48 hrs. (mean ± SEM; n=4 mice/group; triplicate cultures). Two-way ANOVA. **, P
Figure Legend Snippet: Activated B cells show enhanced IL-17 receptor expression (A) Immunohistochemistry was performed on submandibular lymph nodes of naïve and dry eye (DED) mice (21 days after DED induction) to visualize Ki67 (proliferating cells, green), IgM (B cells, red), and IL-17RA (receptor expression, blue) expression. Data are representative of 4–6 mice examined. Images are 10x magnification and 20x magnification for magnified view of areas in white box. (B) Flow cytomteric analysis showing IL-17RA+ CD19+ cells after stimulating purified B cells with medium alone or in the presence or absence of anti-IgM, anti-CD40, and IL-17A for 48 hrs. (mean ± SEM; n=4 mice/group; triplicate cultures). Two-way ANOVA. **, P

Techniques Used: Expressing, Immunohistochemistry, Mouse Assay, Flow Cytometry, Purification

Th17 cells are more effective than Th1 cells to induce B cell proliferation in a contact dependent fashion (A) B cells from the draining lymph nodes (LNs) of dry eye (DED) mice were stimulated with anti-IgM alone or cocultured with anti-CD3 stimulated purified (CD4+CD25−) T effector cells from DED or naïve mice for 48 hrs. The frequencies of proliferating CD19 + B cells were determined by Ki67 staining using flow cytometry (mean± SEM; n=4 mice/group; triplicate cultures). (B) Flow cytometric analysis of Ki67 staining of purified CD19 + B cells from DED mice that were stimulated with anti-IgM alone or cocultured with T cells directly or in transwell inserts with 0.4-μm pores (mean± SEM; n=4 mice/group; triplicate cultures). (C) CD3-stimulated (CD4+CD25−) T effector cell (Teff), IL-17–depleted Teff cells, or IFN-γ–depleted Teff cells were cocultured with IgM-stimulated B cells for 48 hrs. B cell proliferation was assessed with KI67 staining using flow cytomtery. (mean ± SEM; n=6 mice/group; quadruplicate cultures). B cell proliferation is shown as fold change (B cell only [B cell+ anti-IgM] = 1). Data are representative of at least two or more independent experiments. Student’s t test. *, P
Figure Legend Snippet: Th17 cells are more effective than Th1 cells to induce B cell proliferation in a contact dependent fashion (A) B cells from the draining lymph nodes (LNs) of dry eye (DED) mice were stimulated with anti-IgM alone or cocultured with anti-CD3 stimulated purified (CD4+CD25−) T effector cells from DED or naïve mice for 48 hrs. The frequencies of proliferating CD19 + B cells were determined by Ki67 staining using flow cytometry (mean± SEM; n=4 mice/group; triplicate cultures). (B) Flow cytometric analysis of Ki67 staining of purified CD19 + B cells from DED mice that were stimulated with anti-IgM alone or cocultured with T cells directly or in transwell inserts with 0.4-μm pores (mean± SEM; n=4 mice/group; triplicate cultures). (C) CD3-stimulated (CD4+CD25−) T effector cell (Teff), IL-17–depleted Teff cells, or IFN-γ–depleted Teff cells were cocultured with IgM-stimulated B cells for 48 hrs. B cell proliferation was assessed with KI67 staining using flow cytomtery. (mean ± SEM; n=6 mice/group; quadruplicate cultures). B cell proliferation is shown as fold change (B cell only [B cell+ anti-IgM] = 1). Data are representative of at least two or more independent experiments. Student’s t test. *, P

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

37) Product Images from "B Cell Extrinsic Myd88 and Fcer1g Negatively Regulate Autoreactive and Normal B cell Immune Responses"

Article Title: B Cell Extrinsic Myd88 and Fcer1g Negatively Regulate Autoreactive and Normal B cell Immune Responses

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

doi: 10.4049/jimmunol.1600861

FcRγ and MyD88 regulate CD40L dependent cells. BALB/c or MyD88 -deficient Fcer1g -deficient BALB/c mice were sacrificed on day 7 following transfer of purified AM14 sd-Tg B cells, CD40L blocking, and administration of PL2-3 as indicated in Materials and Methods. (A) Surface and intracellular 4-44 double positive cells were quantitated by flow cytometry. (B-C) Splenic 4-44 + AFC of IgM (B) or IgG2a (C) isotype. At least 6 mice per group and 2 independent experiments per time point were compiled. Data are represented as means +/- SEM. *p
Figure Legend Snippet: FcRγ and MyD88 regulate CD40L dependent cells. BALB/c or MyD88 -deficient Fcer1g -deficient BALB/c mice were sacrificed on day 7 following transfer of purified AM14 sd-Tg B cells, CD40L blocking, and administration of PL2-3 as indicated in Materials and Methods. (A) Surface and intracellular 4-44 double positive cells were quantitated by flow cytometry. (B-C) Splenic 4-44 + AFC of IgM (B) or IgG2a (C) isotype. At least 6 mice per group and 2 independent experiments per time point were compiled. Data are represented as means +/- SEM. *p

Techniques Used: Mouse Assay, Purification, Blocking Assay, Flow Cytometry, Cytometry

Contraction of the EF RF response does not require Ccr2. BALB/c or Ccr2 -deficient BALB/c mice were sacrificed on day 7 or 10 following transfer of purified AM14 sd-Tg B cells and administration of PL2-3 as indicated in Materials and Methods. (A) Surface and intracellular 4-44 double positive cells were quantitated by flow cytometry. (B-C) Splenic 4-44 + AFC of IgM (B) or IgG2a (C) isotype. Data are represented as means +/- SEM. At least 5 mice per group and 2 independent experiments per time point were compiled.
Figure Legend Snippet: Contraction of the EF RF response does not require Ccr2. BALB/c or Ccr2 -deficient BALB/c mice were sacrificed on day 7 or 10 following transfer of purified AM14 sd-Tg B cells and administration of PL2-3 as indicated in Materials and Methods. (A) Surface and intracellular 4-44 double positive cells were quantitated by flow cytometry. (B-C) Splenic 4-44 + AFC of IgM (B) or IgG2a (C) isotype. Data are represented as means +/- SEM. At least 5 mice per group and 2 independent experiments per time point were compiled.

Techniques Used: Mouse Assay, Purification, Flow Cytometry, Cytometry

FcRγ and MyD88 regulate T cell dependent antibody production. BALB/c or MyD88 -deficient Fcer1g -deficient BALB/c female BALB/c mice were immunized with NP-CGG in alum one day after adoptive transfer of purified female B1.8 B cells and were sacrificed on day 11 post immunization. Splenic NP2-binding IgM + AFCs (A), NP16-binding IgG1 + AFCs (B) or NP2-binding IgM + AFCs (C) were identified by ELISpot. Two independent experiments with at least 4 mice per group are shown. Data are represented as means +/- SEM. * p
Figure Legend Snippet: FcRγ and MyD88 regulate T cell dependent antibody production. BALB/c or MyD88 -deficient Fcer1g -deficient BALB/c female BALB/c mice were immunized with NP-CGG in alum one day after adoptive transfer of purified female B1.8 B cells and were sacrificed on day 11 post immunization. Splenic NP2-binding IgM + AFCs (A), NP16-binding IgG1 + AFCs (B) or NP2-binding IgM + AFCs (C) were identified by ELISpot. Two independent experiments with at least 4 mice per group are shown. Data are represented as means +/- SEM. * p

Techniques Used: Mouse Assay, Adoptive Transfer Assay, Purification, Binding Assay, Enzyme-linked Immunospot

FcRγ and MyD88 control contraction of the EF RF response. BALB/c or Myd88 -deficient BALB/c or Fcer1g -deficient BALB/c or MyD88 -deficient Fcer1g -deficient BALB/c mice were sacrificed on day 5, 7, or 10 following transfer of purified AM14 sd-Tg B cells and administration of PL2-3 as indicated in Materials and Methods. (A) Representative staining of surface and intracellular 4-44 double positive cells in indicated recipients at d7 following transfer and immunization. (B) Surface and intracellular 4-44 double positive cells as gated in (A) were quantitated by flow cytometry. (C-D) Splenic 4-44 + AFC of IgM (C) or IgG2a (D) isotype were measured by ELISpot. At least 4 mice per group and 2 independent experiments per time point were compiled. Data are represented as means +/- SEM. (E) Expression of indicated activation markers on 4-44 + or 4-44 - B cells at d7 in the indicated recipients was determined by flow cytometry. Statistical comparisons by Mann-Whitney two-tailed test, * p
Figure Legend Snippet: FcRγ and MyD88 control contraction of the EF RF response. BALB/c or Myd88 -deficient BALB/c or Fcer1g -deficient BALB/c or MyD88 -deficient Fcer1g -deficient BALB/c mice were sacrificed on day 5, 7, or 10 following transfer of purified AM14 sd-Tg B cells and administration of PL2-3 as indicated in Materials and Methods. (A) Representative staining of surface and intracellular 4-44 double positive cells in indicated recipients at d7 following transfer and immunization. (B) Surface and intracellular 4-44 double positive cells as gated in (A) were quantitated by flow cytometry. (C-D) Splenic 4-44 + AFC of IgM (C) or IgG2a (D) isotype were measured by ELISpot. At least 4 mice per group and 2 independent experiments per time point were compiled. Data are represented as means +/- SEM. (E) Expression of indicated activation markers on 4-44 + or 4-44 - B cells at d7 in the indicated recipients was determined by flow cytometry. Statistical comparisons by Mann-Whitney two-tailed test, * p

Techniques Used: Mouse Assay, Purification, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunospot, Expressing, Activation Assay, MANN-WHITNEY, Two Tailed Test

38) Product Images from "Growth-factor receptor-bound protein-2 (Grb2) signaling in B cells controls lymphoid follicle organization and germinal center reaction"

Article Title: Growth-factor receptor-bound protein-2 (Grb2) signaling in B cells controls lymphoid follicle organization and germinal center reaction

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

doi: 10.1073/pnas.1016451108

Impairment of GC reaction in Grb2 BKO spleen. Data were obtained from WT and Grb2 BKO mice (8- to 10-wk-old) at day 10 after NP-KLH immunization. ( A ) ( Left ) Contour plots show BrdU + IgD + and BrdU + IgD − populations of the B220 + -gated cells. ( Right ) Frequencies and absolute numbers of BrdU + B cells in WT (white bars) and Grb2 BKO (gray bars) mice are shown as mean with SD ( n = 7). ( B ) Plots are B220 + -gated splenocytes. GC B cells are shown as either CD95 + PNA + population in contour plots or CD95 + GL7 + cells in dot plots. ( C ) Splenocytes were stained with lineage markers (CD4, CD8, Gr1, F4/80), anti-B220, NP, and anti-CD38. ( Upper ) B220 vs. NP staining of gated-lineage marker negative (Lin − ) splenocytes. The percentages of Lin − NP-binding B220 + cells are indicated. The frequencies of IgG1 + CD38 + memory (Mem) and IgG1 + CD38 lo GC subsets within the antigen specific (B220 + NP + ) B-cell compartment are shown in the dot plots ( n = 7). ( D ) Numbers of NP-specific IgM and IgG secreting plasma cells in the spleen of WT (white bars) and Grb2 BKO (gray bars) mice. Each symbol represents a mean value of triplicate samples of an individual mouse.
Figure Legend Snippet: Impairment of GC reaction in Grb2 BKO spleen. Data were obtained from WT and Grb2 BKO mice (8- to 10-wk-old) at day 10 after NP-KLH immunization. ( A ) ( Left ) Contour plots show BrdU + IgD + and BrdU + IgD − populations of the B220 + -gated cells. ( Right ) Frequencies and absolute numbers of BrdU + B cells in WT (white bars) and Grb2 BKO (gray bars) mice are shown as mean with SD ( n = 7). ( B ) Plots are B220 + -gated splenocytes. GC B cells are shown as either CD95 + PNA + population in contour plots or CD95 + GL7 + cells in dot plots. ( C ) Splenocytes were stained with lineage markers (CD4, CD8, Gr1, F4/80), anti-B220, NP, and anti-CD38. ( Upper ) B220 vs. NP staining of gated-lineage marker negative (Lin − ) splenocytes. The percentages of Lin − NP-binding B220 + cells are indicated. The frequencies of IgG1 + CD38 + memory (Mem) and IgG1 + CD38 lo GC subsets within the antigen specific (B220 + NP + ) B-cell compartment are shown in the dot plots ( n = 7). ( D ) Numbers of NP-specific IgM and IgG secreting plasma cells in the spleen of WT (white bars) and Grb2 BKO (gray bars) mice. Each symbol represents a mean value of triplicate samples of an individual mouse.

Techniques Used: Mouse Assay, Staining, Marker, Binding Assay

Altered development and antigen-receptor signaling of grb2 −/− B cells. ( A ) Dot plots show B220 + -gated splenic cells of WT and Grb2 BKO mice. ( Top ) Mature (AA4.1 lo CD24 lo ) and immature (AA4.1 hi CD24 hi ) B cells. ( Middle ) transitional T1 (CD21 hi CD23 hi ) and T2 (CD21 lo CD23 lo ) B cells within the AA4.1 hi CD24 hi -gated immature B-cell population. ( Bottom ) marginal-zone B (CD21 hi CD23 lo ), follicular B (CD21 hi CD23 hi ), and B1 B (CD21 lo CD23 lo ) cells within the AA4.1 lo CD24 lo -gated mature B cells. Percentages of each subset are indicated in the plots. ( B ) Purified B cells (B220 + AA4.1 lo CD24 lo CD21 hi CD23 hi ) were stimulated with anti-IgM F(ab) 2 , anti-CD40, IL-4, and LPS either alone or in combination as indicated. Cell proliferation was measured by 3 H-thymidine incorporation; error bars with SD ( n = 3). ( C ) Intracellular Ca 2+ in B cells was measured by fluorescence intensity of Fluo-4 vs. Fura-red ( n = 8). ( D and E ) Purified B cells from WT or Grb2 BKO mice were stimulated with anti-IgM for various periods. Tyrosine phosphorylation of PLCγ2 and CD22 was determined by immunoprecipitation followed by Western blotting against phosphotyrosine (4G10). Active forms of Erk1/2, JNK, and p38 were directly determined using specific antibodies against individual phosphorylated kinases. The results represent more than three independent experiments.
Figure Legend Snippet: Altered development and antigen-receptor signaling of grb2 −/− B cells. ( A ) Dot plots show B220 + -gated splenic cells of WT and Grb2 BKO mice. ( Top ) Mature (AA4.1 lo CD24 lo ) and immature (AA4.1 hi CD24 hi ) B cells. ( Middle ) transitional T1 (CD21 hi CD23 hi ) and T2 (CD21 lo CD23 lo ) B cells within the AA4.1 hi CD24 hi -gated immature B-cell population. ( Bottom ) marginal-zone B (CD21 hi CD23 lo ), follicular B (CD21 hi CD23 hi ), and B1 B (CD21 lo CD23 lo ) cells within the AA4.1 lo CD24 lo -gated mature B cells. Percentages of each subset are indicated in the plots. ( B ) Purified B cells (B220 + AA4.1 lo CD24 lo CD21 hi CD23 hi ) were stimulated with anti-IgM F(ab) 2 , anti-CD40, IL-4, and LPS either alone or in combination as indicated. Cell proliferation was measured by 3 H-thymidine incorporation; error bars with SD ( n = 3). ( C ) Intracellular Ca 2+ in B cells was measured by fluorescence intensity of Fluo-4 vs. Fura-red ( n = 8). ( D and E ) Purified B cells from WT or Grb2 BKO mice were stimulated with anti-IgM for various periods. Tyrosine phosphorylation of PLCγ2 and CD22 was determined by immunoprecipitation followed by Western blotting against phosphotyrosine (4G10). Active forms of Erk1/2, JNK, and p38 were directly determined using specific antibodies against individual phosphorylated kinases. The results represent more than three independent experiments.

Techniques Used: Mouse Assay, Purification, Fluorescence, Immunoprecipitation, Western Blot

Regulation of CXCR5 signaling and LT expression by Grb2. ( A ) IgM + IgD + naive splenic B cells were purified from WT and Grb2 BKO mice by FACS. Transcripts of LTα, LTβ, and TNF-α were quantified by qRT-PCR and are shown as relative units after being normalized to the expression of β-actin in the corresponding samples; P
Figure Legend Snippet: Regulation of CXCR5 signaling and LT expression by Grb2. ( A ) IgM + IgD + naive splenic B cells were purified from WT and Grb2 BKO mice by FACS. Transcripts of LTα, LTβ, and TNF-α were quantified by qRT-PCR and are shown as relative units after being normalized to the expression of β-actin in the corresponding samples; P

Techniques Used: Expressing, Purification, Mouse Assay, FACS, Quantitative RT-PCR

39) Product Images from "Maternal T cells limit engraftment after in utero hematopoietic cell transplantation in mice"

Article Title: Maternal T cells limit engraftment after in utero hematopoietic cell transplantation in mice

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI44907

The rejection of in utero transplanted allogeneic hematopoietic cells occurs independent of maternal B cells and maternal alloantibodies. ( A ) Frequency of chimerism after IUHCTx of B6 FL cells into fetuses born to a wild-type BALB/c father and either a wild-type BALB/c mother ( n = 43) or a B cell–deficient (JHD) mother ( n = 9). ( B ) Change in levels of chimerism over time in engrafted animals when normalized to the initial level of chimerism at 4 weeks after transplantation (BALB/c mother, n = 10; JHD mother, n = 4). ( C ) Frequency of chimerism in pups fostered by naive mothers (BALB/c fostered, n = 20) and in non-fostered pups (BALB/c, n = 43) after IUHCTx with B6 FL. ( D ) Serum from BALB/c mothers whose fetuses received allogeneic IUHCTx ( n ≥ 10) was analyzed by flow cytometry to quantify total serum IgM (left panels) and IgG (right panels) alloantibody. Comparison groups include naive ( n = 7) and sensitized (Sens., n ≥ 3) mice. ( E ) Total IgM (left panel) and IgG (right panel) alloantibody production at 1, 2, 4, and 6 weeks after sensitization is shown as the MFI relative to a no-serum sample (relative MFI). * P
Figure Legend Snippet: The rejection of in utero transplanted allogeneic hematopoietic cells occurs independent of maternal B cells and maternal alloantibodies. ( A ) Frequency of chimerism after IUHCTx of B6 FL cells into fetuses born to a wild-type BALB/c father and either a wild-type BALB/c mother ( n = 43) or a B cell–deficient (JHD) mother ( n = 9). ( B ) Change in levels of chimerism over time in engrafted animals when normalized to the initial level of chimerism at 4 weeks after transplantation (BALB/c mother, n = 10; JHD mother, n = 4). ( C ) Frequency of chimerism in pups fostered by naive mothers (BALB/c fostered, n = 20) and in non-fostered pups (BALB/c, n = 43) after IUHCTx with B6 FL. ( D ) Serum from BALB/c mothers whose fetuses received allogeneic IUHCTx ( n ≥ 10) was analyzed by flow cytometry to quantify total serum IgM (left panels) and IgG (right panels) alloantibody. Comparison groups include naive ( n = 7) and sensitized (Sens., n ≥ 3) mice. ( E ) Total IgM (left panel) and IgG (right panel) alloantibody production at 1, 2, 4, and 6 weeks after sensitization is shown as the MFI relative to a no-serum sample (relative MFI). * P

Techniques Used: In Utero, Transplantation Assay, Flow Cytometry, Cytometry, Mouse Assay

40) Product Images from "Unperturbed Immune Function despite Mutation of C-Terminal Tyrosines in Syk Previously Implicated in Signaling and Activity Regulation"

Article Title: Unperturbed Immune Function despite Mutation of C-Terminal Tyrosines in Syk Previously Implicated in Signaling and Activity Regulation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00216-17

Normal B cell and T cell development in bone marrow, peritoneal cavity, spleen, and thymus of Syk +/Y3F and Syk Δ/Y3F mice. (A) B cells in the bone marrow (BM) of Syk +/+ , Syk +/Y3F , Syk Δ/+ , and Syk Δ/Y3F ). The bars depict relative frequencies of pre-proB- (B220 hi CD43 + BP1 − CD24 − ) (bars A), early proB- (B220 hi CD43 + BP1 − CD24 + ) (bars B), late pro-/large preB- (B220 hi CD43 + BP1 + CD24 + ) (bars C), small preB- (B220 hi CD43 − IgD − IgM − ) (bars D), immatureB (B220 hi CD43 − IgD + IgM hi ) (bars E), and transitionalB (B220 hi CD43 − IgD + IgM + ) (bars F) cells. (B) Flow cytometric analysis of the mature (B220 + CD23 + CD21 lo ) and marginal zone (B220 + CD23 − CD21 hi ) splenic B cell compartment of Syk +/+ , Syk +/Y3F , Syk Δ/+ , and Syk Δ/Y3F mice. (C) Frequency of proliferating, isolated splenic B cells in response to BCR stimulation. CFSE-labeled B cells were stimulated with 1 μg/ml anti-IgM/anti-CD40 (left) or 10 μg/ml anti-IgM/anti-CD40 (right), and proliferation was determined after 96 h. Stimulation with LPS served as a positive control. The percentage of proliferating cells within the entire population was determined. (D) Flow cytometric analysis of splenic CD4 + CD25 + FoxP3 + regulatory T cells from Syk +/+ , Syk +/Y3F , Syk Δ/+ and Syk Δ/Y3F mice representative of the results of 3 independent experiments. (E) To visualize thymic T cell development, double-negative (CD4 − CD44 − ), DN3 (CD25 + CD44 − ), DN2 (CD25 + CD44 + ), and DN1 (CD25 − CD44 + ) developmental stages are displayed. The numbers indicate relative percentages of total thymic cells. (F) Cell numbers of mature splenic CD3 + and CD3 + CD4 + or CD3 + CD8 + T cells in Syk +/+ , Syk +/Y3F , Syk Δ/+ , and Syk Δ/Y3F mice. **, P ≤ 0.01; ***, P ≤ 0.001. The data are depicted as means and SD.
Figure Legend Snippet: Normal B cell and T cell development in bone marrow, peritoneal cavity, spleen, and thymus of Syk +/Y3F and Syk Δ/Y3F mice. (A) B cells in the bone marrow (BM) of Syk +/+ , Syk +/Y3F , Syk Δ/+ , and Syk Δ/Y3F ). The bars depict relative frequencies of pre-proB- (B220 hi CD43 + BP1 − CD24 − ) (bars A), early proB- (B220 hi CD43 + BP1 − CD24 + ) (bars B), late pro-/large preB- (B220 hi CD43 + BP1 + CD24 + ) (bars C), small preB- (B220 hi CD43 − IgD − IgM − ) (bars D), immatureB (B220 hi CD43 − IgD + IgM hi ) (bars E), and transitionalB (B220 hi CD43 − IgD + IgM + ) (bars F) cells. (B) Flow cytometric analysis of the mature (B220 + CD23 + CD21 lo ) and marginal zone (B220 + CD23 − CD21 hi ) splenic B cell compartment of Syk +/+ , Syk +/Y3F , Syk Δ/+ , and Syk Δ/Y3F mice. (C) Frequency of proliferating, isolated splenic B cells in response to BCR stimulation. CFSE-labeled B cells were stimulated with 1 μg/ml anti-IgM/anti-CD40 (left) or 10 μg/ml anti-IgM/anti-CD40 (right), and proliferation was determined after 96 h. Stimulation with LPS served as a positive control. The percentage of proliferating cells within the entire population was determined. (D) Flow cytometric analysis of splenic CD4 + CD25 + FoxP3 + regulatory T cells from Syk +/+ , Syk +/Y3F , Syk Δ/+ and Syk Δ/Y3F mice representative of the results of 3 independent experiments. (E) To visualize thymic T cell development, double-negative (CD4 − CD44 − ), DN3 (CD25 + CD44 − ), DN2 (CD25 + CD44 + ), and DN1 (CD25 − CD44 + ) developmental stages are displayed. The numbers indicate relative percentages of total thymic cells. (F) Cell numbers of mature splenic CD3 + and CD3 + CD4 + or CD3 + CD8 + T cells in Syk +/+ , Syk +/Y3F , Syk Δ/+ , and Syk Δ/Y3F mice. **, P ≤ 0.01; ***, P ≤ 0.001. The data are depicted as means and SD.

Techniques Used: Mouse Assay, Flow Cytometry, Isolation, Labeling, Positive Control

Syk Y3F transduces indistinguishable BCR-mediated signals in primary B cells and retains the ability to interact with essential BCR downstream signaling components. (A) Primary splenic B cells were either left untreated or stimulated for 3 or 10 min. The cleared cell lysates were used for immunoprecipitation (IP) of Syk (bottom) and subjected to an in vitro kinase assay in the presence of 1 μg recombinant GST-Igβ fusion protein (top). All the blots are representative of the results ≥3 independent experiments. (B to D) Primary splenic B cells isolated from mice of the indicated genotypes were either left untreated or stimulated for 3 or 10 min with 10 μg/ml α-IgM. After cell lysis, successful BCR stimulation was verified in a small sample of total cell lysate (TCL) by anti-pY blotting, and the residual lysate was used for immunoprecipitation. (B) SLP-65 was immunoharvested, and precipitated proteins were analyzed by immunoblotting against phosphotyrosine, SLP-65, and Syk. (C) BCR-associated Igα was immunoprecipitated, and Syk (WT and the Y3F mutant), Vav, and Nck were tested for coprecipitation by Western blotting. (D) Similarly, Syk and NCK were tested for stimulation-dependent interaction with PLCγ. Immunoblots of total cell lysates were probed with antibodies against phosphotyrosine, Syk, and Igα (C) or phosphotyrosine, tubulin, and PLCγ2 (D). Quantification of the data in panels C and D is displayed in Fig. S7 in the supplemental material.
Figure Legend Snippet: Syk Y3F transduces indistinguishable BCR-mediated signals in primary B cells and retains the ability to interact with essential BCR downstream signaling components. (A) Primary splenic B cells were either left untreated or stimulated for 3 or 10 min. The cleared cell lysates were used for immunoprecipitation (IP) of Syk (bottom) and subjected to an in vitro kinase assay in the presence of 1 μg recombinant GST-Igβ fusion protein (top). All the blots are representative of the results ≥3 independent experiments. (B to D) Primary splenic B cells isolated from mice of the indicated genotypes were either left untreated or stimulated for 3 or 10 min with 10 μg/ml α-IgM. After cell lysis, successful BCR stimulation was verified in a small sample of total cell lysate (TCL) by anti-pY blotting, and the residual lysate was used for immunoprecipitation. (B) SLP-65 was immunoharvested, and precipitated proteins were analyzed by immunoblotting against phosphotyrosine, SLP-65, and Syk. (C) BCR-associated Igα was immunoprecipitated, and Syk (WT and the Y3F mutant), Vav, and Nck were tested for coprecipitation by Western blotting. (D) Similarly, Syk and NCK were tested for stimulation-dependent interaction with PLCγ. Immunoblots of total cell lysates were probed with antibodies against phosphotyrosine, Syk, and Igα (C) or phosphotyrosine, tubulin, and PLCγ2 (D). Quantification of the data in panels C and D is displayed in Fig. S7 in the supplemental material.

Techniques Used: Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Isolation, Mouse Assay, Lysis, Mutagenesis, Western Blot

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Software:

Article Title: Essential Immunoregulatory Role for BCAP in B Cell Development and Function
Article Snippet: .. The following mAbs used in the flow cytometric analyses were purchased from BD PharMingen: anti-CD4 (GK1.5), anti-CD5 (53–7.3), anti-CD8 (53–6.72), anti-CD11b (M1/70), anti-CD16 (2.4G2), anti-CD21 (7G6), anti-CD23 (B3B4), anti-CD43 (S7), anti-B220 (RA3–6B2), anti-heat stable antigen (HSA; M1/69), and anti-IgM (R6–60.2), and anti-IgD ( – ) was obtained from Southern Biotechnology Associates, Inc. Data were collected on a FACScan™ flow cytometer (Becton Dickinson) and analyzed using CELLQuest™ software (Becton Dickinson). ..

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  • 86
    SouthernBiotech fluorochrome conjugated anti mouse igm
    Impact of anti-CD20 and/or BAFF blockade on serum immunoglobulin levels in (NZB × NZW)F1 mice. Serum <t>IgM,</t> <t>IgG1,</t> IgG2b, IgA, and IgG3 levels were measured in mice with spontaneous lupus after 6 months of treatment ( A ), mice with IFNα-accelerated lupus after 8 weeks of treatment ( B ), and mice with pristane-accelerated lupus after 12 weeks of treatment ( C ). IgG2a levels were not measured since the treatment reagents were of the mouse IgG2a isotype. Symbols represent individual mice; bars show the mean. ∗ = P
    Fluorochrome Conjugated Anti Mouse Igm, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    SouthernBiotech goat anti murine igm
    Reduction in the B/T cell ratio and reduced levels of total serum antibodies in p52 / p100 (−/−) mice. ( A ) Representative FCM analysis of splenocytes from 16-wk-old p52 / p100 (−/−) and (+/−) mice. <t>IgM–Red</t> 670 versus IgD-FITC two-color profiles are displayed in the top panels. Single-color profiles depict B220-PE and CD3-FITC staining on total splenocytes ( middle and bottom , respectively). Numbers reflect the percentage of positively stained spleen cells. ( B ) Reduced levels of <t>IgG1,</t> IgG2b, and IgA in sera of p52 / p100 (−/−) mice. Titers of total immunoglobulin isotypes from 7–10-wk-old mice of each genotype are shown as indicated. IgG1, IgG2a, and IgA titers differed significantly ( P
    Goat Anti Murine Igm, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 96/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    SouthernBiotech anti igm
    Humoral response of Tssc6 gt/gt mice to immunization with T-cell-dependent and T-cell-independent antigens. The T-cell-dependent antigen NP 18 -KLH was administered i.p. (arrowheads) at the following doses for the primary response. (A) One hundred micrograms of NP 18 -KLH precipitated in alum. (B) Ten micrograms of NP 18 -KLH precipitated in alum. (C) One microgram of NP 18 -KLH precipitated in alum. (D) Twenty micrograms of NP 18 -KLH without adjuvant. Boosting occurred where indicated by the second arrow. (E) Fifty micrograms of NP-LPS injected i.p. on day 0 into each mouse to generate a T-cell-independent immune response. Anti-NP <t>IgM</t> and IgG3 were measured by ELISA at the indicated time points. Data represent the means and standard errors of the means for six mice (panels A through D) or cohorts of three mice (panel E) of each genotype.
    Anti Igm, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 94/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Impact of anti-CD20 and/or BAFF blockade on serum immunoglobulin levels in (NZB × NZW)F1 mice. Serum IgM, IgG1, IgG2b, IgA, and IgG3 levels were measured in mice with spontaneous lupus after 6 months of treatment ( A ), mice with IFNα-accelerated lupus after 8 weeks of treatment ( B ), and mice with pristane-accelerated lupus after 12 weeks of treatment ( C ). IgG2a levels were not measured since the treatment reagents were of the mouse IgG2a isotype. Symbols represent individual mice; bars show the mean. ∗ = P

    Journal: Arthritis & Rheumatology (Hoboken, N.j.)

    Article Title: Dual B Cell Immunotherapy Is Superior to Individual Anti-CD20 Depletion or BAFF Blockade in Murine Models of Spontaneous or Accelerated Lupus

    doi: 10.1002/art.38907

    Figure Lengend Snippet: Impact of anti-CD20 and/or BAFF blockade on serum immunoglobulin levels in (NZB × NZW)F1 mice. Serum IgM, IgG1, IgG2b, IgA, and IgG3 levels were measured in mice with spontaneous lupus after 6 months of treatment ( A ), mice with IFNα-accelerated lupus after 8 weeks of treatment ( B ), and mice with pristane-accelerated lupus after 12 weeks of treatment ( C ). IgG2a levels were not measured since the treatment reagents were of the mouse IgG2a isotype. Symbols represent individual mice; bars show the mean. ∗ = P

    Article Snippet: Fluorochrome-conjugated anti-mouse IgM, IgG1, IgG2a, IgG2b, and IgD antibodies were from SouthernBiotech.

    Techniques: Mouse Assay

    Reduction in the B/T cell ratio and reduced levels of total serum antibodies in p52 / p100 (−/−) mice. ( A ) Representative FCM analysis of splenocytes from 16-wk-old p52 / p100 (−/−) and (+/−) mice. IgM–Red 670 versus IgD-FITC two-color profiles are displayed in the top panels. Single-color profiles depict B220-PE and CD3-FITC staining on total splenocytes ( middle and bottom , respectively). Numbers reflect the percentage of positively stained spleen cells. ( B ) Reduced levels of IgG1, IgG2b, and IgA in sera of p52 / p100 (−/−) mice. Titers of total immunoglobulin isotypes from 7–10-wk-old mice of each genotype are shown as indicated. IgG1, IgG2a, and IgA titers differed significantly ( P

    Journal: The Journal of Experimental Medicine

    Article Title: Mice Deficient in Nuclear Factor (NF)-?B/p52 Present with Defects in Humoral Responses, Germinal Center Reactions, and Splenic Microarchitecture

    doi:

    Figure Lengend Snippet: Reduction in the B/T cell ratio and reduced levels of total serum antibodies in p52 / p100 (−/−) mice. ( A ) Representative FCM analysis of splenocytes from 16-wk-old p52 / p100 (−/−) and (+/−) mice. IgM–Red 670 versus IgD-FITC two-color profiles are displayed in the top panels. Single-color profiles depict B220-PE and CD3-FITC staining on total splenocytes ( middle and bottom , respectively). Numbers reflect the percentage of positively stained spleen cells. ( B ) Reduced levels of IgG1, IgG2b, and IgA in sera of p52 / p100 (−/−) mice. Titers of total immunoglobulin isotypes from 7–10-wk-old mice of each genotype are shown as indicated. IgG1, IgG2a, and IgA titers differed significantly ( P

    Article Snippet: After three washes in PBS-Tween, goat anti–murine IgM, IgG1, IgG2a, IgG2b, and IgG3 conjugated to horseradish peroxidase (HRP; 1:1,000; Southern Biotechnology Assoc.) were added, incubated for 1 h at room temperature, and washed three times with PBS-Tween.

    Techniques: Mouse Assay, Staining

    Defective antibody response to T-dependent antigens in p52-deficient mice. 5–30-wk-old mice of both sexes were injected intraperitoneally with ( A ) 100 μg of TNP (2,4,6 trinitro-phenyl)–KLH (TD), ( B ) 25 μg of TNP-Ficoll (TI-2) or ( C ) 50 μg of TNP-LPS (TI-1). Serum levels of anti-TNP specific antibodies were determined by isotype-specific ELISA 14 d after injection of the TNP conjugates. Antibody levels, expressed as OD, are shown for each genotype, as indicated. Anti-TNP antibodies were undetectable in sera of animals before challenge (data not shown). OD values differed significantly among the following groups of animals by one-way analysis of variance. ( A ) (−/−) versus (+/+) all isotypes tested; (−/−) versus (+/−), IgM, IgG1, IgG2b and IgG3; (+/−) versus (+/+), IgG1, IgG2a, IgG2b. ( C ) (−/−) versus (+/−) and (+/+), IgM, and IgG1. All other groups were not significantly different.

    Journal: The Journal of Experimental Medicine

    Article Title: Mice Deficient in Nuclear Factor (NF)-?B/p52 Present with Defects in Humoral Responses, Germinal Center Reactions, and Splenic Microarchitecture

    doi:

    Figure Lengend Snippet: Defective antibody response to T-dependent antigens in p52-deficient mice. 5–30-wk-old mice of both sexes were injected intraperitoneally with ( A ) 100 μg of TNP (2,4,6 trinitro-phenyl)–KLH (TD), ( B ) 25 μg of TNP-Ficoll (TI-2) or ( C ) 50 μg of TNP-LPS (TI-1). Serum levels of anti-TNP specific antibodies were determined by isotype-specific ELISA 14 d after injection of the TNP conjugates. Antibody levels, expressed as OD, are shown for each genotype, as indicated. Anti-TNP antibodies were undetectable in sera of animals before challenge (data not shown). OD values differed significantly among the following groups of animals by one-way analysis of variance. ( A ) (−/−) versus (+/+) all isotypes tested; (−/−) versus (+/−), IgM, IgG1, IgG2b and IgG3; (+/−) versus (+/+), IgG1, IgG2a, IgG2b. ( C ) (−/−) versus (+/−) and (+/+), IgM, and IgG1. All other groups were not significantly different.

    Article Snippet: After three washes in PBS-Tween, goat anti–murine IgM, IgG1, IgG2a, IgG2b, and IgG3 conjugated to horseradish peroxidase (HRP; 1:1,000; Southern Biotechnology Assoc.) were added, incubated for 1 h at room temperature, and washed three times with PBS-Tween.

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

    Humoral response of Tssc6 gt/gt mice to immunization with T-cell-dependent and T-cell-independent antigens. The T-cell-dependent antigen NP 18 -KLH was administered i.p. (arrowheads) at the following doses for the primary response. (A) One hundred micrograms of NP 18 -KLH precipitated in alum. (B) Ten micrograms of NP 18 -KLH precipitated in alum. (C) One microgram of NP 18 -KLH precipitated in alum. (D) Twenty micrograms of NP 18 -KLH without adjuvant. Boosting occurred where indicated by the second arrow. (E) Fifty micrograms of NP-LPS injected i.p. on day 0 into each mouse to generate a T-cell-independent immune response. Anti-NP IgM and IgG3 were measured by ELISA at the indicated time points. Data represent the means and standard errors of the means for six mice (panels A through D) or cohorts of three mice (panel E) of each genotype.

    Journal: Molecular and Cellular Biology

    Article Title: The Absence of Tssc6, a Member of the Tetraspanin Superfamily, Does Not Affect Lymphoid Development but Enhances In Vitro T-Cell Proliferative Responses

    doi: 10.1128/MCB.22.14.5006-5018.2002

    Figure Lengend Snippet: Humoral response of Tssc6 gt/gt mice to immunization with T-cell-dependent and T-cell-independent antigens. The T-cell-dependent antigen NP 18 -KLH was administered i.p. (arrowheads) at the following doses for the primary response. (A) One hundred micrograms of NP 18 -KLH precipitated in alum. (B) Ten micrograms of NP 18 -KLH precipitated in alum. (C) One microgram of NP 18 -KLH precipitated in alum. (D) Twenty micrograms of NP 18 -KLH without adjuvant. Boosting occurred where indicated by the second arrow. (E) Fifty micrograms of NP-LPS injected i.p. on day 0 into each mouse to generate a T-cell-independent immune response. Anti-NP IgM and IgG3 were measured by ELISA at the indicated time points. Data represent the means and standard errors of the means for six mice (panels A through D) or cohorts of three mice (panel E) of each genotype.

    Article Snippet: Serially diluted serum samples were applied to the washed plates and incubated for at least 4 h. HRP-labeled anti-mouse IgG1 and anti-IgM (Southern Biotechnology Associates, Birmingham, Ala.) at a concentration of 1 μg/ml were applied for 4 h before washing and signal detection by 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate (Sigma).

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

    Flow cytometric analysis of lymphocyte populations in BCAP −/− mice. (A) Expression of BCAP in splenic B cells. Spleen cells stained for CD3 and B220 were analyzed by flow cytometric intracellular staining with an antiserum to BCAP plus goat anti–rabbit IgG-FITC (thick solid line, wild-type mice; shaded area, BCAP −/− mice). Data shown are representative of three independent experiments. (B) Single-cell suspensions from thymus (Thy), bone marrow (BM), lymph node (LN), spleen (SP), and peritoneal cavity (PerC), were stained with the indicated Abs and analyzed using a FACScan ® (wt, wild-type mice). Numbers indicate the percentages of lymphoid cells in the quadrants or enclosed areas. IgM versus IgD and CD21 versus CD23 profiles are shown for B220 + cells, and CD11b versus CD16 profile for B220 − cells. Data shown are representative of six independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Essential Immunoregulatory Role for BCAP in B Cell Development and Function

    doi: 10.1084/jem.20011751

    Figure Lengend Snippet: Flow cytometric analysis of lymphocyte populations in BCAP −/− mice. (A) Expression of BCAP in splenic B cells. Spleen cells stained for CD3 and B220 were analyzed by flow cytometric intracellular staining with an antiserum to BCAP plus goat anti–rabbit IgG-FITC (thick solid line, wild-type mice; shaded area, BCAP −/− mice). Data shown are representative of three independent experiments. (B) Single-cell suspensions from thymus (Thy), bone marrow (BM), lymph node (LN), spleen (SP), and peritoneal cavity (PerC), were stained with the indicated Abs and analyzed using a FACScan ® (wt, wild-type mice). Numbers indicate the percentages of lymphoid cells in the quadrants or enclosed areas. IgM versus IgD and CD21 versus CD23 profiles are shown for B220 + cells, and CD11b versus CD16 profile for B220 − cells. Data shown are representative of six independent experiments.

    Article Snippet: The following mAbs used in the flow cytometric analyses were purchased from BD PharMingen: anti-CD4 (GK1.5), anti-CD5 (53–7.3), anti-CD8 (53–6.72), anti-CD11b (M1/70), anti-CD16 (2.4G2), anti-CD21 (7G6), anti-CD23 (B3B4), anti-CD43 (S7), anti-B220 (RA3–6B2), anti-heat stable antigen (HSA; M1/69), and anti-IgM (R6–60.2), and anti-IgD ( – ) was obtained from Southern Biotechnology Associates, Inc. Data were collected on a FACScan™ flow cytometer (Becton Dickinson) and analyzed using CELLQuest™ software (Becton Dickinson).

    Techniques: Flow Cytometry, Mouse Assay, Expressing, Staining

    Impaired proliferative response of BCAP −/− splenic B cells. (A) Purified splenic B cells from wild-type and BCAP −/− mice were cultured with medium, F(ab′) 2 goat anti-IgM Ab (15 μg/ml), rat anti-CD40 Ab (10 μg/ml), or LPS (10 μg/ml). The mean and standard deviations are plotted for wild-type (black bars) and BCAP −/− splenic B cells (white bars). Experiments were performed in triplicates. Data shown are representative of three independent experiments. (B) Splenic B cells from the indicated mice were sorted into B220 + HSA hi (immature B) and B220 + HSA lo (mature B) subsets, and experiments were performed in triplicates. Data shown are representative of three independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Essential Immunoregulatory Role for BCAP in B Cell Development and Function

    doi: 10.1084/jem.20011751

    Figure Lengend Snippet: Impaired proliferative response of BCAP −/− splenic B cells. (A) Purified splenic B cells from wild-type and BCAP −/− mice were cultured with medium, F(ab′) 2 goat anti-IgM Ab (15 μg/ml), rat anti-CD40 Ab (10 μg/ml), or LPS (10 μg/ml). The mean and standard deviations are plotted for wild-type (black bars) and BCAP −/− splenic B cells (white bars). Experiments were performed in triplicates. Data shown are representative of three independent experiments. (B) Splenic B cells from the indicated mice were sorted into B220 + HSA hi (immature B) and B220 + HSA lo (mature B) subsets, and experiments were performed in triplicates. Data shown are representative of three independent experiments.

    Article Snippet: The following mAbs used in the flow cytometric analyses were purchased from BD PharMingen: anti-CD4 (GK1.5), anti-CD5 (53–7.3), anti-CD8 (53–6.72), anti-CD11b (M1/70), anti-CD16 (2.4G2), anti-CD21 (7G6), anti-CD23 (B3B4), anti-CD43 (S7), anti-B220 (RA3–6B2), anti-heat stable antigen (HSA; M1/69), and anti-IgM (R6–60.2), and anti-IgD ( – ) was obtained from Southern Biotechnology Associates, Inc. Data were collected on a FACScan™ flow cytometer (Becton Dickinson) and analyzed using CELLQuest™ software (Becton Dickinson).

    Techniques: Purification, Mouse Assay, Cell Culture