Structured Review

Becton Dickinson anti igm
Immunoglobulin expression is needed for ER maintenance in resting B cells. (A) Mature resting B cells from B1-8 flox (B18f) and B1-8 flox × R26Stop flox P110* (B18f P110*) mice were treated with TAT-Cre to induce the deletion of loxP-flanked sequences. The frequency of BCR-negative B cells was monitored over time. (B) Cell size of B1-8 flox × Mb1-creERT2 B cells treated with tamoxifen to induce the deletion of the Ig heavy chain. (B, C) Cell size (B) and ER mass (C) of cells positive for surface <t>IgM</t> (IgM+) and negative for surface IgM (IgM−) was determined on day 3. Data were tested for significance using the paired t test. (A, B) N = 3; P = 0.75 (A), ** P = 0.0087 (B). Experiments were performed in six technical replicates. Circles indicate mean values of these replicates obtained from three different mice. (D) Mature resting B cells from B1-8 flox × EµBcl2 mice treated with TAT-Cre to induce the deletion of the Ig heavy chain, FACS-sorted on d5 upon TAT-Cre, and let to recover for 24 h post-sorting were resuspended in medium containing pyruvate, glutamine, and glucose, and OCR was measured using Seahorse flux technology on d6. The measurements were performed in technical triplicates and are displayed as mean ± SD. a, oligomycin; b, FCCP; c, rotenone + antimycin. (E) mRNA levels for the indicated genes were determined on day 2 after TAT-Cre–mediated deletion of loxP-flanked sequences in B18f and B18f P110* B cells positive or negative for surface IgM expression. Results were tested for significance using two-way ANOVA. For clarity, only differences between IgM+/IgM− and IgM+ P110*/IgM− P110* cells are shown. ** P = 0.0078 and 0.0057, * P = 0.0351 and 0.0402. Graph shows data obtained from three mice. (F, G, H) B cells from B1-8 flox × EµBcl2 mice were purified, treated with TAT-Cre, kept for 4–6 d in culture to allow for IgM deletion, and stimulated overnight with LPS. (F, G, H) BiP protein levels (F), cell size (G), and <t>CD69</t> surface levels (H) were assessed by flow cytometry. Results were tested for significance using ANOVA. For clarity only differences between IgM+ and IgM− cells are shown. Circles represent data obtained from different mice. If samples were measured in technical replicates, circles represent mean values obtained from these replicates. For F: N = 7, ** P = 0.002; for G: N = 4, *** P = 0.0006; for H: N = 5, * P = 0.0192. Heavy chain deletion was performed in an in vitro cell culture in the presence of the cytokine BAFF.
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1) Product Images from "Immunoglobulin expression in the endoplasmic reticulum shapes the metabolic fitness of B lymphocytes"

Article Title: Immunoglobulin expression in the endoplasmic reticulum shapes the metabolic fitness of B lymphocytes

Journal: Life Science Alliance

doi: 10.26508/lsa.202000700

Immunoglobulin expression is needed for ER maintenance in resting B cells. (A) Mature resting B cells from B1-8 flox (B18f) and B1-8 flox × R26Stop flox P110* (B18f P110*) mice were treated with TAT-Cre to induce the deletion of loxP-flanked sequences. The frequency of BCR-negative B cells was monitored over time. (B) Cell size of B1-8 flox × Mb1-creERT2 B cells treated with tamoxifen to induce the deletion of the Ig heavy chain. (B, C) Cell size (B) and ER mass (C) of cells positive for surface IgM (IgM+) and negative for surface IgM (IgM−) was determined on day 3. Data were tested for significance using the paired t test. (A, B) N = 3; P = 0.75 (A), ** P = 0.0087 (B). Experiments were performed in six technical replicates. Circles indicate mean values of these replicates obtained from three different mice. (D) Mature resting B cells from B1-8 flox × EµBcl2 mice treated with TAT-Cre to induce the deletion of the Ig heavy chain, FACS-sorted on d5 upon TAT-Cre, and let to recover for 24 h post-sorting were resuspended in medium containing pyruvate, glutamine, and glucose, and OCR was measured using Seahorse flux technology on d6. The measurements were performed in technical triplicates and are displayed as mean ± SD. a, oligomycin; b, FCCP; c, rotenone + antimycin. (E) mRNA levels for the indicated genes were determined on day 2 after TAT-Cre–mediated deletion of loxP-flanked sequences in B18f and B18f P110* B cells positive or negative for surface IgM expression. Results were tested for significance using two-way ANOVA. For clarity, only differences between IgM+/IgM− and IgM+ P110*/IgM− P110* cells are shown. ** P = 0.0078 and 0.0057, * P = 0.0351 and 0.0402. Graph shows data obtained from three mice. (F, G, H) B cells from B1-8 flox × EµBcl2 mice were purified, treated with TAT-Cre, kept for 4–6 d in culture to allow for IgM deletion, and stimulated overnight with LPS. (F, G, H) BiP protein levels (F), cell size (G), and CD69 surface levels (H) were assessed by flow cytometry. Results were tested for significance using ANOVA. For clarity only differences between IgM+ and IgM− cells are shown. Circles represent data obtained from different mice. If samples were measured in technical replicates, circles represent mean values obtained from these replicates. For F: N = 7, ** P = 0.002; for G: N = 4, *** P = 0.0006; for H: N = 5, * P = 0.0192. Heavy chain deletion was performed in an in vitro cell culture in the presence of the cytokine BAFF.
Figure Legend Snippet: Immunoglobulin expression is needed for ER maintenance in resting B cells. (A) Mature resting B cells from B1-8 flox (B18f) and B1-8 flox × R26Stop flox P110* (B18f P110*) mice were treated with TAT-Cre to induce the deletion of loxP-flanked sequences. The frequency of BCR-negative B cells was monitored over time. (B) Cell size of B1-8 flox × Mb1-creERT2 B cells treated with tamoxifen to induce the deletion of the Ig heavy chain. (B, C) Cell size (B) and ER mass (C) of cells positive for surface IgM (IgM+) and negative for surface IgM (IgM−) was determined on day 3. Data were tested for significance using the paired t test. (A, B) N = 3; P = 0.75 (A), ** P = 0.0087 (B). Experiments were performed in six technical replicates. Circles indicate mean values of these replicates obtained from three different mice. (D) Mature resting B cells from B1-8 flox × EµBcl2 mice treated with TAT-Cre to induce the deletion of the Ig heavy chain, FACS-sorted on d5 upon TAT-Cre, and let to recover for 24 h post-sorting were resuspended in medium containing pyruvate, glutamine, and glucose, and OCR was measured using Seahorse flux technology on d6. The measurements were performed in technical triplicates and are displayed as mean ± SD. a, oligomycin; b, FCCP; c, rotenone + antimycin. (E) mRNA levels for the indicated genes were determined on day 2 after TAT-Cre–mediated deletion of loxP-flanked sequences in B18f and B18f P110* B cells positive or negative for surface IgM expression. Results were tested for significance using two-way ANOVA. For clarity, only differences between IgM+/IgM− and IgM+ P110*/IgM− P110* cells are shown. ** P = 0.0078 and 0.0057, * P = 0.0351 and 0.0402. Graph shows data obtained from three mice. (F, G, H) B cells from B1-8 flox × EµBcl2 mice were purified, treated with TAT-Cre, kept for 4–6 d in culture to allow for IgM deletion, and stimulated overnight with LPS. (F, G, H) BiP protein levels (F), cell size (G), and CD69 surface levels (H) were assessed by flow cytometry. Results were tested for significance using ANOVA. For clarity only differences between IgM+ and IgM− cells are shown. Circles represent data obtained from different mice. If samples were measured in technical replicates, circles represent mean values obtained from these replicates. For F: N = 7, ** P = 0.002; for G: N = 4, *** P = 0.0006; for H: N = 5, * P = 0.0192. Heavy chain deletion was performed in an in vitro cell culture in the presence of the cytokine BAFF.

Techniques Used: Expressing, Mouse Assay, FACS, Purification, Flow Cytometry, In Vitro, Cell Culture

BCR expression boosts B lymphoma fitness but is not absolutely required for survival. (A) Cells were stained with anti-IgM and anti-IgD to examine cell surface expression of the BCR. Anti-CD19 was used as a control. (B) Expression of the μ-heavy chain, λ-light chain, Igα, and Igβ were determined by Western blot. One of three independent experiments is shown. (C) The cells were plated on d0 and cell numbers were assessed on d1, d2, and d3 using the CCK8-kit. Values were normalized to the measurement obtained on d1. Significance was determined using the ANOVA test. (D) Expression of the indicated proteins was determined by Western blot. One of four independent experiments is shown. (E) Forward scatter (FSC-A) as a measure of cell size was determined using flow cytometry. Significance was determined using the Mann–Whitney test. N = 8. (F) Biological triplicates of Ig heavy chain–deficient DG75 cells were sorted on d3 upon Ig heavy chain deletion and pooled for analysis. Expression of the Ig heavy chain (antibody directed against the CH domain) and histone 3 (H3) was determined by Western blot. One of two independent experiments is shown. (G) Cell size measured on d6 after Ig heavy chain deletion. Significance was determined using the paired t test. N = 3; ** P = 0.0074. (H) Relative abundance of WT and H-KO DG75 cells in a mixed culture at the indicated time points after Ig heavy chain deletion. Linear regression analysis was performed. Slopes of the WT and KO abundance lines were found to be significantly different. P
Figure Legend Snippet: BCR expression boosts B lymphoma fitness but is not absolutely required for survival. (A) Cells were stained with anti-IgM and anti-IgD to examine cell surface expression of the BCR. Anti-CD19 was used as a control. (B) Expression of the μ-heavy chain, λ-light chain, Igα, and Igβ were determined by Western blot. One of three independent experiments is shown. (C) The cells were plated on d0 and cell numbers were assessed on d1, d2, and d3 using the CCK8-kit. Values were normalized to the measurement obtained on d1. Significance was determined using the ANOVA test. (D) Expression of the indicated proteins was determined by Western blot. One of four independent experiments is shown. (E) Forward scatter (FSC-A) as a measure of cell size was determined using flow cytometry. Significance was determined using the Mann–Whitney test. N = 8. (F) Biological triplicates of Ig heavy chain–deficient DG75 cells were sorted on d3 upon Ig heavy chain deletion and pooled for analysis. Expression of the Ig heavy chain (antibody directed against the CH domain) and histone 3 (H3) was determined by Western blot. One of two independent experiments is shown. (G) Cell size measured on d6 after Ig heavy chain deletion. Significance was determined using the paired t test. N = 3; ** P = 0.0074. (H) Relative abundance of WT and H-KO DG75 cells in a mixed culture at the indicated time points after Ig heavy chain deletion. Linear regression analysis was performed. Slopes of the WT and KO abundance lines were found to be significantly different. P

Techniques Used: Expressing, Staining, Western Blot, Flow Cytometry, MANN-WHITNEY

2) Product Images from "IL-4 upregulates Igα and Igβ protein, resulting in augmented IgM maturation and BCR-triggered B cell activation"

Article Title: IL-4 upregulates Igα and Igβ protein, resulting in augmented IgM maturation and BCR-triggered B cell activation

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

doi: 10.4049/jimmunol.1203211

IL-4 promotes IgM maturation. Purified splenic B cells were cultured in medium for 3 h (MED, fine line) or treated with IL-4 (bold line) for 24 hours and subjected to flow cytometric analysis using fluorescent monoclonal antibody against IgM (A), IgD
Figure Legend Snippet: IL-4 promotes IgM maturation. Purified splenic B cells were cultured in medium for 3 h (MED, fine line) or treated with IL-4 (bold line) for 24 hours and subjected to flow cytometric analysis using fluorescent monoclonal antibody against IgM (A), IgD

Techniques Used: Purification, Cell Culture, Flow Cytometry

3) Product Images from "The PI3K Isoforms p110α and p110δ are Essential for Pre-B Cell Receptor Signaling and B Cell Development"

Article Title: The PI3K Isoforms p110α and p110δ are Essential for Pre-B Cell Receptor Signaling and B Cell Development

Journal: Science signaling

doi: 10.1126/scisignal.2001104

Intact PI3K signaling in B cells from CD2Cre-p110α fl mice. ( A ) PCR analysis of genomic DNA from purified B cells. The 812 kb band is from a recombined allele, whereas the 591 bp band is from an allele with both loxP sites intact. ( B ) Western blotting analysis of p110α, p110β, p110δ, and p85 from B cells purified from wild-type, CD2Cre-p110α fl , and CD2Cre-p110β fl mice. ( C ) Western blotting analysis of B cells stimulated with antibody against IgM F(ab’) 2 for 3, 10, or 30 min. Blots were incubated with antibodies against pAkt (Ser 407 ), pFoxo1 (Thr 24 )/Foxo3(Thr 32 ), pERK (Ser 202 Tyr 204 ), and total Akt. The cells in the panel on the right had been treated with IC87114 (5 μM). These data are representative of three experiments. ( D ) Proliferation of CD2Cre-p110α fl B cells in response to antibody against IgM F(ab’)2 (αIgM, 10 μg/ml), antibody against CD40 (αCD40,10 μg/ml), IL-4 (20 ng/ml), antibody against CD40 with IL-4, or LPS (10 μg/ml). The cpm represents the amount of 3 H thymidine incorporated during the last 6 hours of the 48 hours of culture. The mean values represent averages from 6 mice analyzed in 3 independent experiments. The error bars represent the standard error of the mean (SEM). ( E ) Specific IgG 1 titers after primary and secondary challenge. p110α fl (filled circles) and CD2Cre-p110α fl (open circles) mice were immunized with TNP-KLH on days 0 and day 292 and bled on days 7, 14, 292, and 302. EC 50 values were calculated from serially diluted serum samples on TNP-BSA–coated plates. Each dot represents EC 50 IgG 1 values obtained from one of six mice per group.
Figure Legend Snippet: Intact PI3K signaling in B cells from CD2Cre-p110α fl mice. ( A ) PCR analysis of genomic DNA from purified B cells. The 812 kb band is from a recombined allele, whereas the 591 bp band is from an allele with both loxP sites intact. ( B ) Western blotting analysis of p110α, p110β, p110δ, and p85 from B cells purified from wild-type, CD2Cre-p110α fl , and CD2Cre-p110β fl mice. ( C ) Western blotting analysis of B cells stimulated with antibody against IgM F(ab’) 2 for 3, 10, or 30 min. Blots were incubated with antibodies against pAkt (Ser 407 ), pFoxo1 (Thr 24 )/Foxo3(Thr 32 ), pERK (Ser 202 Tyr 204 ), and total Akt. The cells in the panel on the right had been treated with IC87114 (5 μM). These data are representative of three experiments. ( D ) Proliferation of CD2Cre-p110α fl B cells in response to antibody against IgM F(ab’)2 (αIgM, 10 μg/ml), antibody against CD40 (αCD40,10 μg/ml), IL-4 (20 ng/ml), antibody against CD40 with IL-4, or LPS (10 μg/ml). The cpm represents the amount of 3 H thymidine incorporated during the last 6 hours of the 48 hours of culture. The mean values represent averages from 6 mice analyzed in 3 independent experiments. The error bars represent the standard error of the mean (SEM). ( E ) Specific IgG 1 titers after primary and secondary challenge. p110α fl (filled circles) and CD2Cre-p110α fl (open circles) mice were immunized with TNP-KLH on days 0 and day 292 and bled on days 7, 14, 292, and 302. EC 50 values were calculated from serially diluted serum samples on TNP-BSA–coated plates. Each dot represents EC 50 IgG 1 values obtained from one of six mice per group.

Techniques Used: Mouse Assay, Polymerase Chain Reaction, Purification, Western Blot, Incubation

4) Product Images from "B Cells in Murine Cervical Lymph Nodes are Conventional B-2 Cells"

Article Title: B Cells in Murine Cervical Lymph Nodes are Conventional B-2 Cells

Journal: Journal of Korean Medical Science

doi: 10.3346/jkms.2006.21.3.391

Antigen surface expression on ( A ) cervical lymph node ( B ) spleen ( C ) peritoneal fluid B cells. B cells of BALB/cByJ mice were harvested and stained with Abs specific for B220, CD5, CD23, CD43, B7.1, B7.2, IgM, and Syndacan-1. Expression of functional molecules on B cells was analyzed by two-color flow cytometry.
Figure Legend Snippet: Antigen surface expression on ( A ) cervical lymph node ( B ) spleen ( C ) peritoneal fluid B cells. B cells of BALB/cByJ mice were harvested and stained with Abs specific for B220, CD5, CD23, CD43, B7.1, B7.2, IgM, and Syndacan-1. Expression of functional molecules on B cells was analyzed by two-color flow cytometry.

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

5) Product Images from "Memory B cells, but not long-lived plasma cells, possess antigen specificities for viral escape mutants"

Article Title: Memory B cells, but not long-lived plasma cells, possess antigen specificities for viral escape mutants

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20110740

MBCs can respond to and neutralize variant virus. (A) Neutralization curves of WNV-WT or WNV-K307E with supernatant from stimulated MBCs. Data reflects four independent experiments performed in duplicate. Error bars indicate SD. (B) IgH a MBCs (CD19 + IgM − IgD − lin − CCR6 + CD80 + ) from WNV-vaccinated mice were sorted and transferred into allotypic IgH b recipients and challenged with WNV-WT or WNV-K307E 1 d later. Specificity of the MBC-derived antibody at different time points was determined by ELISA using an IgG2a a -specific detection antibody. Data reflects eight mice per group and is the average of five experiments (C) Mice were immunized with JEV-DIII and treated with B cell–depleting (CD20 mAb) or an isotype control antibody. Serum was tested before WNV infection (left) and at days 3 and 8 (right) after infection for IgG binding to WNV-DIII by ELISA. The data reflect a total of five to nine mice per group from three experiments. P-values were determined using an unpaired, two-tailed Student’s t test (*, P
Figure Legend Snippet: MBCs can respond to and neutralize variant virus. (A) Neutralization curves of WNV-WT or WNV-K307E with supernatant from stimulated MBCs. Data reflects four independent experiments performed in duplicate. Error bars indicate SD. (B) IgH a MBCs (CD19 + IgM − IgD − lin − CCR6 + CD80 + ) from WNV-vaccinated mice were sorted and transferred into allotypic IgH b recipients and challenged with WNV-WT or WNV-K307E 1 d later. Specificity of the MBC-derived antibody at different time points was determined by ELISA using an IgG2a a -specific detection antibody. Data reflects eight mice per group and is the average of five experiments (C) Mice were immunized with JEV-DIII and treated with B cell–depleting (CD20 mAb) or an isotype control antibody. Serum was tested before WNV infection (left) and at days 3 and 8 (right) after infection for IgG binding to WNV-DIII by ELISA. The data reflect a total of five to nine mice per group from three experiments. P-values were determined using an unpaired, two-tailed Student’s t test (*, P

Techniques Used: Variant Assay, Neutralization, Mouse Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Infection, Binding Assay, Two Tailed Test

6) Product Images from "CEACAM1 induces B-cell survival and is essential for protective antiviral antibody production"

Article Title: CEACAM1 induces B-cell survival and is essential for protective antiviral antibody production

Journal: Nature Communications

doi: 10.1038/ncomms7217

CEACAM1 in B cells induces survival genes via Syk and Erk and NF-κB. ( a , b ) Representative immunoblot probed with antibodies to phospho-Igα, Igα and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in wild-type (WT) and Ceacam1 −/− splenocytes ( a , left panel) or in WT Splenocytes ( a , right panel) or probed with antibodies to phospho-Syk, Syk and GAPDH in WT and Ceacam1 −/− splenocytes ( b , left panel) or in WT splenocytes ( b , right panel) after treatment with anti-IgM or anti-CEACAM1 monoclonal antibody or LPS for indicated time points ( n =4). ( c ) Representative flow cytometry histogram of WT or Ceacam1 −/− splenocytes gated on B cells showing p-Btk (pY223)/Itk (pY180) staining; splenocytes were left untreated (grey area) or were treated with anti-IgM or anti-CEACAM1 antibody for indicated time points at 37 °C ( n =3). ( d , e ) Representative immunoblot probed with antibodies to Tyr-P-Erk (p-p44/42 MAPK), Erk (p44/42) and GAPDH ( d , n =4) or with antibodies to phospho-p38, p38 and GAPDH ( e , n =3) in WT splenocytes after treatment with anti-CEACAM1 antibody or LPS for indicated time points. ( f ) Representative immunoblot and quantification of specific bands after staining for p-NF-κB p65, NF-κB p65, IκBα and β-actin in WT or Ceacam1 −/− B cells after challenge with anti-CEACAM1 antibody for indicated time points ( n =4). ( g – k ) RT–PCR analysis of representative transcription factors such as Bcl-6 , Pax-5 ( g , n =5), Bcl2a1 , Xiap ( g , n =7), NF-κB p65, Rel-B ( h , n =5–7), Nfatc1 , Nfatc2 ( i , n =5) or c-Jun , c-Fos , and Ap1s1 ( j , n =5) or Blimp1 ( k , n =7) mRNA from WT and Ceacam1 −/− B cells sorted from spleen and 24 h after without stimulation (values show fold change to expression in B cells 0 h after sorting). *P
Figure Legend Snippet: CEACAM1 in B cells induces survival genes via Syk and Erk and NF-κB. ( a , b ) Representative immunoblot probed with antibodies to phospho-Igα, Igα and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in wild-type (WT) and Ceacam1 −/− splenocytes ( a , left panel) or in WT Splenocytes ( a , right panel) or probed with antibodies to phospho-Syk, Syk and GAPDH in WT and Ceacam1 −/− splenocytes ( b , left panel) or in WT splenocytes ( b , right panel) after treatment with anti-IgM or anti-CEACAM1 monoclonal antibody or LPS for indicated time points ( n =4). ( c ) Representative flow cytometry histogram of WT or Ceacam1 −/− splenocytes gated on B cells showing p-Btk (pY223)/Itk (pY180) staining; splenocytes were left untreated (grey area) or were treated with anti-IgM or anti-CEACAM1 antibody for indicated time points at 37 °C ( n =3). ( d , e ) Representative immunoblot probed with antibodies to Tyr-P-Erk (p-p44/42 MAPK), Erk (p44/42) and GAPDH ( d , n =4) or with antibodies to phospho-p38, p38 and GAPDH ( e , n =3) in WT splenocytes after treatment with anti-CEACAM1 antibody or LPS for indicated time points. ( f ) Representative immunoblot and quantification of specific bands after staining for p-NF-κB p65, NF-κB p65, IκBα and β-actin in WT or Ceacam1 −/− B cells after challenge with anti-CEACAM1 antibody for indicated time points ( n =4). ( g – k ) RT–PCR analysis of representative transcription factors such as Bcl-6 , Pax-5 ( g , n =5), Bcl2a1 , Xiap ( g , n =7), NF-κB p65, Rel-B ( h , n =5–7), Nfatc1 , Nfatc2 ( i , n =5) or c-Jun , c-Fos , and Ap1s1 ( j , n =5) or Blimp1 ( k , n =7) mRNA from WT and Ceacam1 −/− B cells sorted from spleen and 24 h after without stimulation (values show fold change to expression in B cells 0 h after sorting). *P

Techniques Used: Flow Cytometry, Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing

CEACAM1 is expressed on murine B-cell subsets. ( a , b ) Representative histogram showing CEACAM1 expression in leukocyte subpopulations isolated from wild-type (WT, black line) and Ceacam1 −/− mice (red line). Isotype control antibody staining of leukocytes from WT mice is shown as a grey area. Peripheral blood leukocytes gated for Ly6G (granulocytes) and CD115 (monocytes; a ) and CD90.2 + (T cells) or B220 + (B cells) cells ( b ), as measured by flow cytometry ( n =6 per group). ( c – e ) Dot plot showing IgD and IgM expression of cells gated on B220, and histogram showing CEACAM1 expression (black line) versus isotype control antibody staining (grey area) in bone marrow ( c ), lymph nodes ( d ) and spleens ( e ), respectively, from WT mice, as measured by flow cytometry ( n =6 per group). ( f ) Representative expression levels of CEACAM1-S and CEACAM1-L isoforms in murine splenic B cells by real-time PCR ( n =6). ** P
Figure Legend Snippet: CEACAM1 is expressed on murine B-cell subsets. ( a , b ) Representative histogram showing CEACAM1 expression in leukocyte subpopulations isolated from wild-type (WT, black line) and Ceacam1 −/− mice (red line). Isotype control antibody staining of leukocytes from WT mice is shown as a grey area. Peripheral blood leukocytes gated for Ly6G (granulocytes) and CD115 (monocytes; a ) and CD90.2 + (T cells) or B220 + (B cells) cells ( b ), as measured by flow cytometry ( n =6 per group). ( c – e ) Dot plot showing IgD and IgM expression of cells gated on B220, and histogram showing CEACAM1 expression (black line) versus isotype control antibody staining (grey area) in bone marrow ( c ), lymph nodes ( d ) and spleens ( e ), respectively, from WT mice, as measured by flow cytometry ( n =6 per group). ( f ) Representative expression levels of CEACAM1-S and CEACAM1-L isoforms in murine splenic B cells by real-time PCR ( n =6). ** P

Techniques Used: Expressing, Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction

7) Product Images from "The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain"

Article Title: The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20121069

B cell maturation pathways and B cell function are impaired in SPPL2a −/− mice. (A) BM from either wild-type or SPPL2a-deficient mice was transplanted into irradiated Rag2 −/− cγc −/− mice. After 10 wk, B cell subsets in BM and spleen were analyzed by co-staining of B220 together with IgM or CD21 and CD24, respectively, and quantified as percentage of viable cells (numbers). Data shown are representative of eight experiments ( Table 3 ). (B) B220 + cells were isolated from spleens of SPPL2a +/+ and SPPL2a −/− mice and stained for CD21, CD24, and BAFF-R. Histograms show the BAFF-R expression on SPPL2a +/+ (solid line, black) and SPPL2a −/− (solid line, red) T1 B cells (B220 + CD21 low CD24 high ) from a representative of three independent experiments. Specificity of the BAFF-R staining was confirmed by the respective isotype controls (dashed lines). (C) Splenocytes from SPPL2a −/− and wild-type mice were stained for B220, CD21, and CD24 and loaded with the ratiometric Ca 2+ -sensitive fluorophore Indo-1-AM. After monitoring of basal Ca 2+ concentrations in T1 B cells (B220 + CD21 low CD24 high ) for 30 s, cells were stimulated with goat anti–mouse IgM F(ab’) 2 fragments and Ca 2+ flux was recorded for 5 min in the absence of extracellular Ca 2+ and for an additional 5 min in the presence of 1 mM extracellular CaCl 2 . Data are representative of three experiments and were also confirmed in repopulated RAG −/− cγc −/− mice (not depicted). (D) Plasma immunoglobulin levels were measured in wild-type ( wt ) and SPPL2a −/− mice ( ko ). Mean ± SD; n = 8–12. ***, P
Figure Legend Snippet: B cell maturation pathways and B cell function are impaired in SPPL2a −/− mice. (A) BM from either wild-type or SPPL2a-deficient mice was transplanted into irradiated Rag2 −/− cγc −/− mice. After 10 wk, B cell subsets in BM and spleen were analyzed by co-staining of B220 together with IgM or CD21 and CD24, respectively, and quantified as percentage of viable cells (numbers). Data shown are representative of eight experiments ( Table 3 ). (B) B220 + cells were isolated from spleens of SPPL2a +/+ and SPPL2a −/− mice and stained for CD21, CD24, and BAFF-R. Histograms show the BAFF-R expression on SPPL2a +/+ (solid line, black) and SPPL2a −/− (solid line, red) T1 B cells (B220 + CD21 low CD24 high ) from a representative of three independent experiments. Specificity of the BAFF-R staining was confirmed by the respective isotype controls (dashed lines). (C) Splenocytes from SPPL2a −/− and wild-type mice were stained for B220, CD21, and CD24 and loaded with the ratiometric Ca 2+ -sensitive fluorophore Indo-1-AM. After monitoring of basal Ca 2+ concentrations in T1 B cells (B220 + CD21 low CD24 high ) for 30 s, cells were stimulated with goat anti–mouse IgM F(ab’) 2 fragments and Ca 2+ flux was recorded for 5 min in the absence of extracellular Ca 2+ and for an additional 5 min in the presence of 1 mM extracellular CaCl 2 . Data are representative of three experiments and were also confirmed in repopulated RAG −/− cγc −/− mice (not depicted). (D) Plasma immunoglobulin levels were measured in wild-type ( wt ) and SPPL2a −/− mice ( ko ). Mean ± SD; n = 8–12. ***, P

Techniques Used: Cell Function Assay, Mouse Assay, Irradiation, Staining, Isolation, Expressing

Disturbance of membrane traffic within the endocytic system of SPPL2a −/− B cells. (A and B) Visualization of CD74 in isolated splenic SPPL2a +/+ and SPPL2a −/− B cells by indirect immunofluorescence using an antibody against an N-terminal epitope detecting the NTF and the full-length protein. EEA1 (A) and LAMP1 (B) served as markers of early endosomes and lysosomes/late endosomes, respectively. Bars, 2 µm. (C) Transmission electron microscopy of splenic IgM + B cells from wild-type or SPPL2a −/− mice. Bars, 1 µm. (D) Vacuoles in SPPL2a −/− B cells exhibited various contents of low-electron density. Occasionally, multivesicular bodies (mvb) were observed (arrow). Bar, 500 nm. (E) Presence of CD74 NTF, LAMP1, and MHCII in vacuoles of IgM + B cells from SPPL2a −/− mice was assessed by immunogold labeling. Bars, 200 nm (CD74 and MHCII single labeling) or 100 nm (CD74 + LAMP1 double labeling). (F and G) Surface and total MHCII levels in transitional stage T1 B cells (B220 + CD21 low CD24 high ) of SPPL2a-deficient or wild-type mice. Splenocytes were stained for B220, CD21, and CD24, allowing for identification of B cell subsets. Subsequently, cells were incubated with anti-MHCII with or without previous permeabilization and analyzed by flow cytometry. Surface and total MHCII levels are shown as histograms representative of three independent experiments or as mean of median fluorescence intensity (MFI) from three mice per genotype (G). ***, P
Figure Legend Snippet: Disturbance of membrane traffic within the endocytic system of SPPL2a −/− B cells. (A and B) Visualization of CD74 in isolated splenic SPPL2a +/+ and SPPL2a −/− B cells by indirect immunofluorescence using an antibody against an N-terminal epitope detecting the NTF and the full-length protein. EEA1 (A) and LAMP1 (B) served as markers of early endosomes and lysosomes/late endosomes, respectively. Bars, 2 µm. (C) Transmission electron microscopy of splenic IgM + B cells from wild-type or SPPL2a −/− mice. Bars, 1 µm. (D) Vacuoles in SPPL2a −/− B cells exhibited various contents of low-electron density. Occasionally, multivesicular bodies (mvb) were observed (arrow). Bar, 500 nm. (E) Presence of CD74 NTF, LAMP1, and MHCII in vacuoles of IgM + B cells from SPPL2a −/− mice was assessed by immunogold labeling. Bars, 200 nm (CD74 and MHCII single labeling) or 100 nm (CD74 + LAMP1 double labeling). (F and G) Surface and total MHCII levels in transitional stage T1 B cells (B220 + CD21 low CD24 high ) of SPPL2a-deficient or wild-type mice. Splenocytes were stained for B220, CD21, and CD24, allowing for identification of B cell subsets. Subsequently, cells were incubated with anti-MHCII with or without previous permeabilization and analyzed by flow cytometry. Surface and total MHCII levels are shown as histograms representative of three independent experiments or as mean of median fluorescence intensity (MFI) from three mice per genotype (G). ***, P

Techniques Used: Isolation, Immunofluorescence, Transmission Assay, Electron Microscopy, Mouse Assay, Labeling, Staining, Incubation, Flow Cytometry, Cytometry, Fluorescence

8) Product Images from "Regulation and Maintenance of an Adoptive T-Cell Dependent Memory B Cell Pool"

Article Title: Regulation and Maintenance of an Adoptive T-Cell Dependent Memory B Cell Pool

Journal: PLoS ONE

doi: 10.1371/journal.pone.0167003

Development of B cell primary response upon adoptive transfer of SW HEL .AID/YFP.Rag2 -/- B cells. (A) Rag2-/- hosts were injected intravenously with naive SW HEL .AID/YFP.Rag2 -/- B cells together with OTII.Rag2 -/- naive CD4 T cells. Recipient mice were immunized 24 hours later and B cell response analyzed 7, 14 days and 8 weeks after. (B) Flow cytometric analyses of specific HEL + CD19 + (left panels) splenic B cells from SW HEL .AID/YFP.Rag2 -/- naive donor (upper panels), immunized mice reconstituted only with B cells (middle panels) and immunized mice reconstituted with both B and T cells 14 days after immunization (lower panels), for the expression of YFP (middle panels), and IgM and IgG (right panels). (C) Number of splenic B cells recovered from the recipient mice (left panel) and repartition of AID/YFP- and AID/YFP+ among splenic B cells (right panel) 7, 11 and 14 days after immunization. (D) Seric levels of anti-HEL specific IgG (left panel) and IgM (right panel) in immunized recipients 7, 11 and 14 days after immunization. (E) AID/YFP (green), B220 (red), IgD (blue) (right panels) and merged ( left and right panels) expression by confocal microscopy analysis of spleen slices 14 days after immunization. (F) Analysis of germinal center B cells in immunized mice. Left panel shows the co-expression of Gl7 and CD95 by B cells from naive (left dot plot), non-immunized (middle dot plot) and 14 day-immunized mice (right dot plot). Right panel shows the % of Gl7hi CD95+ cells among AID/YFP- (white bar) and AID/YFP+ (black bar) splenic B cell subsets from 14 day-immunized mice. Data (mean ± SEM) are shown for one experiment representative of 3, with 4–5 mice per group. Significances were calculated using Student t -tests, *, P
Figure Legend Snippet: Development of B cell primary response upon adoptive transfer of SW HEL .AID/YFP.Rag2 -/- B cells. (A) Rag2-/- hosts were injected intravenously with naive SW HEL .AID/YFP.Rag2 -/- B cells together with OTII.Rag2 -/- naive CD4 T cells. Recipient mice were immunized 24 hours later and B cell response analyzed 7, 14 days and 8 weeks after. (B) Flow cytometric analyses of specific HEL + CD19 + (left panels) splenic B cells from SW HEL .AID/YFP.Rag2 -/- naive donor (upper panels), immunized mice reconstituted only with B cells (middle panels) and immunized mice reconstituted with both B and T cells 14 days after immunization (lower panels), for the expression of YFP (middle panels), and IgM and IgG (right panels). (C) Number of splenic B cells recovered from the recipient mice (left panel) and repartition of AID/YFP- and AID/YFP+ among splenic B cells (right panel) 7, 11 and 14 days after immunization. (D) Seric levels of anti-HEL specific IgG (left panel) and IgM (right panel) in immunized recipients 7, 11 and 14 days after immunization. (E) AID/YFP (green), B220 (red), IgD (blue) (right panels) and merged ( left and right panels) expression by confocal microscopy analysis of spleen slices 14 days after immunization. (F) Analysis of germinal center B cells in immunized mice. Left panel shows the co-expression of Gl7 and CD95 by B cells from naive (left dot plot), non-immunized (middle dot plot) and 14 day-immunized mice (right dot plot). Right panel shows the % of Gl7hi CD95+ cells among AID/YFP- (white bar) and AID/YFP+ (black bar) splenic B cell subsets from 14 day-immunized mice. Data (mean ± SEM) are shown for one experiment representative of 3, with 4–5 mice per group. Significances were calculated using Student t -tests, *, P

Techniques Used: Adoptive Transfer Assay, Injection, Mouse Assay, Flow Cytometry, Expressing, Confocal Microscopy

Functions of AID/YFP+ memory B cell subsets. Rag2-/- recipient mice were injected with naive SW HEL .AID/YFP.Rag2 -/- B cells and OTII.Rag2-/- naive CD4 T cells and immunized as in Fig 1 . Eight weeks after immunization, AID/YFP+ IgM+ and AID/YFP+ IgG+ subsets were isolated and transferred alone into secondary Rag2-/- recipient mice. Naive B cells were transferred as controls. (A) Numbers of splenic B cells recovered from the different secondary hosts without immunization. (B) The secondary hosts were immunized 1 day after transfer and the seric levels of anti-HEL specific IgG (C) and IgM were measured 6, 9, 12, 16, 20 and 30 days after immunization. (D) the % of splenic Gl7 hi B cells in secondary hosts transferred with YFP+ IgM+ (white) or YFP+ IgG+ (black) B cells was analyzed by Flow Cytometry 3 weeks after immunization and (F) the relative % of AID/YFP- and AID/YFP+ cells was assessed. In another settings, (G) the different secondary hosts were immunized either 1 day (black circles) or 30 days (white circles) after transfer and the number of total splenic B cells recovered from the spleen of different secondary hosts was determined 6 weeks after immunization. Data (mean ± SEM) are shown for one experiment representative of 2, with 4–5 mice per group. Significances were calculated using Student t -tests, *, P
Figure Legend Snippet: Functions of AID/YFP+ memory B cell subsets. Rag2-/- recipient mice were injected with naive SW HEL .AID/YFP.Rag2 -/- B cells and OTII.Rag2-/- naive CD4 T cells and immunized as in Fig 1 . Eight weeks after immunization, AID/YFP+ IgM+ and AID/YFP+ IgG+ subsets were isolated and transferred alone into secondary Rag2-/- recipient mice. Naive B cells were transferred as controls. (A) Numbers of splenic B cells recovered from the different secondary hosts without immunization. (B) The secondary hosts were immunized 1 day after transfer and the seric levels of anti-HEL specific IgG (C) and IgM were measured 6, 9, 12, 16, 20 and 30 days after immunization. (D) the % of splenic Gl7 hi B cells in secondary hosts transferred with YFP+ IgM+ (white) or YFP+ IgG+ (black) B cells was analyzed by Flow Cytometry 3 weeks after immunization and (F) the relative % of AID/YFP- and AID/YFP+ cells was assessed. In another settings, (G) the different secondary hosts were immunized either 1 day (black circles) or 30 days (white circles) after transfer and the number of total splenic B cells recovered from the spleen of different secondary hosts was determined 6 weeks after immunization. Data (mean ± SEM) are shown for one experiment representative of 2, with 4–5 mice per group. Significances were calculated using Student t -tests, *, P

Techniques Used: Mouse Assay, Injection, Isolation, Flow Cytometry, Cytometry

Long-term maintenance of memory B cells. Rag2-/- recipient mice were injected with naive SW HEL .AID/YFP.Rag2 -/- B cells and OTII.Rag2-/- naive CD4 T cells and immunized as in Fig 1 . Mice were analyzed at 2, 4, 8, 12 and 20 weeks after immunization. (A) Number of splenic B cells recovered. (B) Seric level of anti-HEL specific IgG1. (C) Number of splenic CD138+ plasma cells determined by flow cytometry. (D) Repartition of splenic AID/YFP+ (black bars) and AID/YFP- (white bars) B cells. (E) Flow cytometric analyses of IgG and IgM expression by AID/YFP+ B cells. (F) Flow cytometric analyses of MHC II, CD80, CD95, CXCR5 and Gl7 expression by splenic AID/YFP+ (green lines), AID/YFP- (blue lines) and naive (black lines, upper histograms), and IgM+ (black lines) and IgG+ (red lines, lower histograms) B cells from naive, and eight-week immunized mice. (G) The % of splenic proliferating B cell was assessed 8 weeks after immunization by flow cytometric analysis of Ki67 expression (left) and BrdU incorporation 24h (middle) or 72h (right) after BrdU injection. (H) The % of apoptotic splenic B cell was assessed by flow cytometric analysis of active caspase 3 level 8 weeks after immunization. Data (mean ± SEM) are shown for one experiment representative of 2, with 4–5 mice per group. Significances were calculated using Student t -tests, *, P
Figure Legend Snippet: Long-term maintenance of memory B cells. Rag2-/- recipient mice were injected with naive SW HEL .AID/YFP.Rag2 -/- B cells and OTII.Rag2-/- naive CD4 T cells and immunized as in Fig 1 . Mice were analyzed at 2, 4, 8, 12 and 20 weeks after immunization. (A) Number of splenic B cells recovered. (B) Seric level of anti-HEL specific IgG1. (C) Number of splenic CD138+ plasma cells determined by flow cytometry. (D) Repartition of splenic AID/YFP+ (black bars) and AID/YFP- (white bars) B cells. (E) Flow cytometric analyses of IgG and IgM expression by AID/YFP+ B cells. (F) Flow cytometric analyses of MHC II, CD80, CD95, CXCR5 and Gl7 expression by splenic AID/YFP+ (green lines), AID/YFP- (blue lines) and naive (black lines, upper histograms), and IgM+ (black lines) and IgG+ (red lines, lower histograms) B cells from naive, and eight-week immunized mice. (G) The % of splenic proliferating B cell was assessed 8 weeks after immunization by flow cytometric analysis of Ki67 expression (left) and BrdU incorporation 24h (middle) or 72h (right) after BrdU injection. (H) The % of apoptotic splenic B cell was assessed by flow cytometric analysis of active caspase 3 level 8 weeks after immunization. Data (mean ± SEM) are shown for one experiment representative of 2, with 4–5 mice per group. Significances were calculated using Student t -tests, *, P

Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry, Expressing, BrdU Incorporation Assay

9) Product Images from "Sequential immunization induces strong and broad immunity against all four dengue virus serotypes"

Article Title: Sequential immunization induces strong and broad immunity against all four dengue virus serotypes

Journal: NPJ Vaccines

doi: 10.1038/s41541-020-00216-0

Comparison of VH gene usages by two strategies at different dosage of immunization. a Heatmap shows the V H family usages by clone frequencies with red to blue to gray corresponding to from high to low to absence. Shot indicates the order of immunization and isotype indicates IgD, IgM and IgG. b Comparison of IGHV5 family gene usage frequency between tetravalent and sequential immunizations. The Chi-square test was performed for statistical analysis.
Figure Legend Snippet: Comparison of VH gene usages by two strategies at different dosage of immunization. a Heatmap shows the V H family usages by clone frequencies with red to blue to gray corresponding to from high to low to absence. Shot indicates the order of immunization and isotype indicates IgD, IgM and IgG. b Comparison of IGHV5 family gene usage frequency between tetravalent and sequential immunizations. The Chi-square test was performed for statistical analysis.

Techniques Used:

Comparison of antigen-specific B-cell responses in tetravalent and sequential immunized mice. Two weeks after last immunization, splenocytes were stained with Alexa conjugated DENV1 and DENV2 E proteins and appropriate antibodies (see list in “Methods”). The antigen-specific DENV1 + (left panel), DENV2 + (middle panel) and DENV1 + DENV2 + (right panel) B cells ( a ), IgG1 + B cells ( b ), IgM + IgD + B cells ( c ), and germinal center B cells ( d ), and their corresponding Bcl6 expression ( e ) were assessed. Each dot represents one mouse and mean ± SD are shown. The p -values (ns: not significant ( p > 0.05), * p
Figure Legend Snippet: Comparison of antigen-specific B-cell responses in tetravalent and sequential immunized mice. Two weeks after last immunization, splenocytes were stained with Alexa conjugated DENV1 and DENV2 E proteins and appropriate antibodies (see list in “Methods”). The antigen-specific DENV1 + (left panel), DENV2 + (middle panel) and DENV1 + DENV2 + (right panel) B cells ( a ), IgG1 + B cells ( b ), IgM + IgD + B cells ( c ), and germinal center B cells ( d ), and their corresponding Bcl6 expression ( e ) were assessed. Each dot represents one mouse and mean ± SD are shown. The p -values (ns: not significant ( p > 0.05), * p

Techniques Used: Mouse Assay, Staining, Expressing

10) Product Images from "Homeostatically proliferating CD4+ T cells are involved in the pathogenesis of an Omenn syndrome murine model"

Article Title: Homeostatically proliferating CD4+ T cells are involved in the pathogenesis of an Omenn syndrome murine model

Journal:

doi: 10.1172/JCI30513

MM have high serum concentrations of IgE, IgM, IgG, and IgA.
Figure Legend Snippet: MM have high serum concentrations of IgE, IgM, IgG, and IgA.

Techniques Used:

11) Product Images from "Specific NEMO mutations impair CD40-mediated c-Rel activation and B cell terminal differentiation"

Article Title: Specific NEMO mutations impair CD40-mediated c-Rel activation and B cell terminal differentiation

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI200421345

XHM-ED B cells have a specific CD40 pathway proliferation defect. CD19 + B cells from XHM-ED patients and normal controls were cultured in triplicate for 72 hours with IL-4 (gray bars), CD40L + IL-4 (black bars), or goat anti-human IgM Fab + IL-4 (white bars). Data are the mean ± SD proliferative response and are representative of 3 experiments.
Figure Legend Snippet: XHM-ED B cells have a specific CD40 pathway proliferation defect. CD19 + B cells from XHM-ED patients and normal controls were cultured in triplicate for 72 hours with IL-4 (gray bars), CD40L + IL-4 (black bars), or goat anti-human IgM Fab + IL-4 (white bars). Data are the mean ± SD proliferative response and are representative of 3 experiments.

Techniques Used: Cell Culture

12) Product Images from "Subnuclear cyclin D3 compartments and the coordinated regulation of proliferation and immunoglobulin variable gene repression"

Article Title: Subnuclear cyclin D3 compartments and the coordinated regulation of proliferation and immunoglobulin variable gene repression

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20120800

Alteration of PI3K signaling does not affect B cell progenitor proliferation. (A) B cell progenitors from WT and Bim −/− bone marrow were cultured in the presence of IL-7 with or without LY294002 for 24 h. Cells were then fixed and analyzed for DNA content and the percentage of cells in G 1 versus SG 2 M phase of cell cycle are provided. Data are representative of three independent experiments. (B) B cell progenitors from Bim −/− bone marrow were cultured in the presence of IL-7 with or without LY294002 for 24 h. TCLs were prepared in NP-40 lysis buffer, resolved by SDS-PAGE, and membranes probed for cyclin D3 and Actin. Relative molecular mass (kD) is as indicated. Data are representative of three independent experiments. (C) Flow cytometry for expression of B220, CD43, and IgM by bone marrow cells from p85α +/− and p85α −/− mice. Numbers in contour plots indicate the percentage of cells in each gate. Representative data, n = 8. (D) Relative number of cells in each population determined using total number of bone marrow cells after red blood cell lysis and percentages from flow cytometry. Each point represents a single mouse and bars represent the mean for each population. (E) The corresponding populations from C were analyzed for DNA content. Numbers in histogram plots indicate the percentage of cells in G 1 and SG 2 M phases of cell cycle. (F) Summary of the percentage of cells in SG 2 M from each population. Each point represents a single mouse and bars represent the mean for each population. (G and H) bPTEN +/+ and bPTEN −/− bone marrow was analyzed as in C and D (G), and the percentage of cells in SG 2 M from each population was summarized (H), n = 4. Each point represents a single mouse and bars represent the mean for each population. *, P
Figure Legend Snippet: Alteration of PI3K signaling does not affect B cell progenitor proliferation. (A) B cell progenitors from WT and Bim −/− bone marrow were cultured in the presence of IL-7 with or without LY294002 for 24 h. Cells were then fixed and analyzed for DNA content and the percentage of cells in G 1 versus SG 2 M phase of cell cycle are provided. Data are representative of three independent experiments. (B) B cell progenitors from Bim −/− bone marrow were cultured in the presence of IL-7 with or without LY294002 for 24 h. TCLs were prepared in NP-40 lysis buffer, resolved by SDS-PAGE, and membranes probed for cyclin D3 and Actin. Relative molecular mass (kD) is as indicated. Data are representative of three independent experiments. (C) Flow cytometry for expression of B220, CD43, and IgM by bone marrow cells from p85α +/− and p85α −/− mice. Numbers in contour plots indicate the percentage of cells in each gate. Representative data, n = 8. (D) Relative number of cells in each population determined using total number of bone marrow cells after red blood cell lysis and percentages from flow cytometry. Each point represents a single mouse and bars represent the mean for each population. (E) The corresponding populations from C were analyzed for DNA content. Numbers in histogram plots indicate the percentage of cells in G 1 and SG 2 M phases of cell cycle. (F) Summary of the percentage of cells in SG 2 M from each population. Each point represents a single mouse and bars represent the mean for each population. (G and H) bPTEN +/+ and bPTEN −/− bone marrow was analyzed as in C and D (G), and the percentage of cells in SG 2 M from each population was summarized (H), n = 4. Each point represents a single mouse and bars represent the mean for each population. *, P

Techniques Used: Cell Culture, Lysis, SDS Page, Flow Cytometry, Cytometry, Expressing, Mouse Assay

13) Product Images from "In vivo conditional deletion of HDAC7 reveals its requirement to establish proper B lymphocyte identity and development"

Article Title: In vivo conditional deletion of HDAC7 reveals its requirement to establish proper B lymphocyte identity and development

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20150821

HDAC7 is required for early B cell development. (A) Absolute numbers of total bone marrow cells ( Hdac7 fl/− [ n = 6] and Hdac7 +/− [ n = 8]) and total spleen cells ( Hdac7 +/− [ n = 12] and Hdac7 fl/− [ n = 9]). (B) Representative photograph of the spleen from Hdac7 +/− and Hdac7 fl/− mice. (C) Absolute numbers of bone marrow B220 + B cells ( Hdac7 fl/− [ n = 5] and Hdac7 +/− [ n = 8]), B220 + CD43 + IgM − pro–B cells ( Hdac7 fl/− [ n = 5] and Hdac7 +/− [ n = 8]), B220 + CD43 − IgM − pre–B cells ( Hdac7 fl/− [ n = 6] and Hdac7 +/− [ n = 6]), B220 + IgM + immature B cells ( Hdac7 fl/− [ n = 7] and Hdac7 +/− [ n = 9]), and B220 + IgM + IgD + mature recirculating B cells ( Hdac7 fl/− [ n = 7] and Hdac7 +/− [ n = 9]). (D) Graph on the left shows the absolute numbers of B220 + B cells from spleen of Hdac7 +/− ( n = 12) and Hdac7 fl/− ( n = 9) mice. Graph in the middle shows the percentage of B220 + cells in the blood from Hdac7 +/− ( n = 4) and Hdac7 fl/− ( n = 4) mice. Graph on the right shows absolute numbers of spleen B cell subsets from wild-type and HDAC7-deficient mice: B220 + IgM + IgD + mature B cells ( Hdac7 +/− [ n = 7] and Hdac7 fl/− [ n = 5]), CD21 bright CD23 + marginal zone (MZ) B cells ( Hdac7 +/− [ n = 12] and Hdac7 fl/− [ n = 9]), and CD21 + CD23 bright CD93 − follicular (FO; Hdac7 +/− [ n = 9] and Hdac7 fl/− [ n = 7]) and CD21 + CD23 bright CD93 + transitional (T; Hdac7 +/− [ n = 8] and Hdac7 fl/− [ n = 7]) B cells. (E) Hematoxylin and eosin staining of the spleen from Hdac7 +/− and Hdac7 fl/− mice. (F) Percentages of granulocytes and macrophages from the spleen of Hdac7 +/− ( n = 9) and Hdac7 fl/− ( n = 7) mice. Mean values are shown as horizontal bars. ns, not significant. (G) Representative FACS analyses from three independent experiments are shown on T lymphocytes from the spleens of Hdac7 +/− and Hdac7 fl/− mice. FSC, forward scatter. Data are represented as the mean ± SEM. Statistical significances were identified using the unpaired two-tailed Student’s t test. *, P
Figure Legend Snippet: HDAC7 is required for early B cell development. (A) Absolute numbers of total bone marrow cells ( Hdac7 fl/− [ n = 6] and Hdac7 +/− [ n = 8]) and total spleen cells ( Hdac7 +/− [ n = 12] and Hdac7 fl/− [ n = 9]). (B) Representative photograph of the spleen from Hdac7 +/− and Hdac7 fl/− mice. (C) Absolute numbers of bone marrow B220 + B cells ( Hdac7 fl/− [ n = 5] and Hdac7 +/− [ n = 8]), B220 + CD43 + IgM − pro–B cells ( Hdac7 fl/− [ n = 5] and Hdac7 +/− [ n = 8]), B220 + CD43 − IgM − pre–B cells ( Hdac7 fl/− [ n = 6] and Hdac7 +/− [ n = 6]), B220 + IgM + immature B cells ( Hdac7 fl/− [ n = 7] and Hdac7 +/− [ n = 9]), and B220 + IgM + IgD + mature recirculating B cells ( Hdac7 fl/− [ n = 7] and Hdac7 +/− [ n = 9]). (D) Graph on the left shows the absolute numbers of B220 + B cells from spleen of Hdac7 +/− ( n = 12) and Hdac7 fl/− ( n = 9) mice. Graph in the middle shows the percentage of B220 + cells in the blood from Hdac7 +/− ( n = 4) and Hdac7 fl/− ( n = 4) mice. Graph on the right shows absolute numbers of spleen B cell subsets from wild-type and HDAC7-deficient mice: B220 + IgM + IgD + mature B cells ( Hdac7 +/− [ n = 7] and Hdac7 fl/− [ n = 5]), CD21 bright CD23 + marginal zone (MZ) B cells ( Hdac7 +/− [ n = 12] and Hdac7 fl/− [ n = 9]), and CD21 + CD23 bright CD93 − follicular (FO; Hdac7 +/− [ n = 9] and Hdac7 fl/− [ n = 7]) and CD21 + CD23 bright CD93 + transitional (T; Hdac7 +/− [ n = 8] and Hdac7 fl/− [ n = 7]) B cells. (E) Hematoxylin and eosin staining of the spleen from Hdac7 +/− and Hdac7 fl/− mice. (F) Percentages of granulocytes and macrophages from the spleen of Hdac7 +/− ( n = 9) and Hdac7 fl/− ( n = 7) mice. Mean values are shown as horizontal bars. ns, not significant. (G) Representative FACS analyses from three independent experiments are shown on T lymphocytes from the spleens of Hdac7 +/− and Hdac7 fl/− mice. FSC, forward scatter. Data are represented as the mean ± SEM. Statistical significances were identified using the unpaired two-tailed Student’s t test. *, P

Techniques Used: Mouse Assay, Staining, FACS, Two Tailed Test

14) Product Images from "The E3 ubiquitin ligase Mule acts through the ATM-p53 axis to maintain B lymphocyte homeostasis"

Article Title: The E3 ubiquitin ligase Mule acts through the ATM-p53 axis to maintain B lymphocyte homeostasis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20111363

Impaired proliferation and activation of BMKO B cells. (A) Littermate control and BMKO mice were supplied with BrdU-containing drinking water for 3.5 d. Top left, total splenic B cells were stained with anti-BrdU and anti-B220 Abs and analyzed by flow cytometry ( n = 5; **, P = 0.0021). Bottom left, gated B220 + IgM + cells were further gated on CD93 + and sorted to detect BrdU + TS B cells ( n = 4; *, P = 0.024). Top right, B220 + IgM + cells were gated on CD21 + CD23 low and sorted to detect BrdU + MZ B cells ( n = 4; ***, P = 0.000077). Bottom right, B220 + IgM + cells were gated on CD21 + CD23 + and sorted for BrdU + FO B cells ( n = 4; ***, P = 0.0001). Numbers are the percentage of BrdU + cells among the gated population. (B) Purified splenic littermate control and BMKO B cells were stimulated for 24 h with the indicated doses of anti-IgM (left) or anti-IgM plus anti-CD40 (right) and incubated with [ 3 H]-thymidine. Results are the mean ± SD of triplicate. (C) Purified splenic littermate control and BMKO B cells were stimulated with 10 µg/ml anti-IgM or 10 µg/ml anti-IgM plus 0.5 µg/ml CD40 ligand for 24 h. Up-regulation of the indicated activation markers was monitored by flow cytometry. (D) Purified splenic littermate control and BMKO B cells were stimulated with 20 µg/ml anti-IgM for the indicated times. Lysates were immunoblotted to detect phospho-tyrosine (pTyr). (E) Splenic littermate control and BMKO B cells preloaded with Indo-1 were stimulated first with anti-IgM and then with thapsigargin (TG; positive control). The Ca 2+ response was measured based on the ratio of violet (Ca 2+ bound) to blue (Ca 2+ free) fluorescence. Results (A–E) are representative of two to three independent experiments involving one to five mice per genotype.
Figure Legend Snippet: Impaired proliferation and activation of BMKO B cells. (A) Littermate control and BMKO mice were supplied with BrdU-containing drinking water for 3.5 d. Top left, total splenic B cells were stained with anti-BrdU and anti-B220 Abs and analyzed by flow cytometry ( n = 5; **, P = 0.0021). Bottom left, gated B220 + IgM + cells were further gated on CD93 + and sorted to detect BrdU + TS B cells ( n = 4; *, P = 0.024). Top right, B220 + IgM + cells were gated on CD21 + CD23 low and sorted to detect BrdU + MZ B cells ( n = 4; ***, P = 0.000077). Bottom right, B220 + IgM + cells were gated on CD21 + CD23 + and sorted for BrdU + FO B cells ( n = 4; ***, P = 0.0001). Numbers are the percentage of BrdU + cells among the gated population. (B) Purified splenic littermate control and BMKO B cells were stimulated for 24 h with the indicated doses of anti-IgM (left) or anti-IgM plus anti-CD40 (right) and incubated with [ 3 H]-thymidine. Results are the mean ± SD of triplicate. (C) Purified splenic littermate control and BMKO B cells were stimulated with 10 µg/ml anti-IgM or 10 µg/ml anti-IgM plus 0.5 µg/ml CD40 ligand for 24 h. Up-regulation of the indicated activation markers was monitored by flow cytometry. (D) Purified splenic littermate control and BMKO B cells were stimulated with 20 µg/ml anti-IgM for the indicated times. Lysates were immunoblotted to detect phospho-tyrosine (pTyr). (E) Splenic littermate control and BMKO B cells preloaded with Indo-1 were stimulated first with anti-IgM and then with thapsigargin (TG; positive control). The Ca 2+ response was measured based on the ratio of violet (Ca 2+ bound) to blue (Ca 2+ free) fluorescence. Results (A–E) are representative of two to three independent experiments involving one to five mice per genotype.

Techniques Used: Activation Assay, Mouse Assay, Staining, Flow Cytometry, Cytometry, Purification, Incubation, Positive Control, Fluorescence

15) Product Images from "B Cell Activating Factor Inhibition Impairs Bacterial Immunity by Reducing T Cell-Independent IgM Secretion"

Article Title: B Cell Activating Factor Inhibition Impairs Bacterial Immunity by Reducing T Cell-Independent IgM Secretion

Journal: Infection and Immunity

doi: 10.1128/IAI.00998-13

BAFF signaling regulates the expression of CD138, but not that of other cell surface molecules, on TI plasmablasts. (A) B220 low CD11c low plasmablasts from the spleens of control and 10F4-treated mice were analyzed for surface expression of IgM and CD138
Figure Legend Snippet: BAFF signaling regulates the expression of CD138, but not that of other cell surface molecules, on TI plasmablasts. (A) B220 low CD11c low plasmablasts from the spleens of control and 10F4-treated mice were analyzed for surface expression of IgM and CD138

Techniques Used: Expressing, Mouse Assay

BAFF signaling is required for the maintenance of IgM production during chronic infection. (A) A BAFF-neutralizing antibody was administered on days 63, 70, and 77 postinfection, and bone marrow cells were analyzed for surface expression of IgM and CD138
Figure Legend Snippet: BAFF signaling is required for the maintenance of IgM production during chronic infection. (A) A BAFF-neutralizing antibody was administered on days 63, 70, and 77 postinfection, and bone marrow cells were analyzed for surface expression of IgM and CD138

Techniques Used: Infection, Expressing

16) Product Images from "S1P1 receptor directs the release of immature B cells from bone marrow into blood"

Article Title: S1P1 receptor directs the release of immature B cells from bone marrow into blood

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20092210

Impaired release of immature bone marrow B cells by CXCR4 antagonism in the absence of S1P1 receptor. (A) Mice were injected with the CXCR4 antagonist AMD3100 or vehicle alone, and bone marrow and blood were collected after 90 min. Immature bone marrow B cells in the blood (B220 + CD93 + ) were gated as IgM + IgD − and IgM + IgD low , and quantified from control and B- S1pr1 KO mice. Results are shown as absolute numbers in 250 µl of blood. Bars represent mean values, and the closed circles are individual mice. Data are representative of three experiments. *, P
Figure Legend Snippet: Impaired release of immature bone marrow B cells by CXCR4 antagonism in the absence of S1P1 receptor. (A) Mice were injected with the CXCR4 antagonist AMD3100 or vehicle alone, and bone marrow and blood were collected after 90 min. Immature bone marrow B cells in the blood (B220 + CD93 + ) were gated as IgM + IgD − and IgM + IgD low , and quantified from control and B- S1pr1 KO mice. Results are shown as absolute numbers in 250 µl of blood. Bars represent mean values, and the closed circles are individual mice. Data are representative of three experiments. *, P

Techniques Used: Mouse Assay, Injection

Migration of bone marrow B cells toward S1P is mediated by the S1P3 receptor. (A) Total bone marrow cells were added to a Transwell insert and allowed to respond to increasing concentrations of S1P or to 100 ng/ml SDF-1 in the lower well. Percentages of the input that were found in the lower well after a 3-h incubation with S1P (left axis) or with SDF-1 (right axis) were plotted for pro–/pre– (B220 + IgD − IgM − ), immature (B220 + IgM + IgD − and B220 + IgM + IgD low ), and mature (B220 + IgM + IgD high ) B cells. Data are presented as mean values ± SD ( n = 3 for each genotype) and are representative of three independent experiments. *, P
Figure Legend Snippet: Migration of bone marrow B cells toward S1P is mediated by the S1P3 receptor. (A) Total bone marrow cells were added to a Transwell insert and allowed to respond to increasing concentrations of S1P or to 100 ng/ml SDF-1 in the lower well. Percentages of the input that were found in the lower well after a 3-h incubation with S1P (left axis) or with SDF-1 (right axis) were plotted for pro–/pre– (B220 + IgD − IgM − ), immature (B220 + IgM + IgD − and B220 + IgM + IgD low ), and mature (B220 + IgM + IgD high ) B cells. Data are presented as mean values ± SD ( n = 3 for each genotype) and are representative of three independent experiments. *, P

Techniques Used: Migration, Incubation

Reduced numbers of mature and immature B cells in the blood of B- S1pr1 KO mice. (A–D) Bone marrow B cell subpopulations from control and B- S1pr1 KO mice were analyzed by flow cytometry using FITC-conjugated anti-B220, APC-conjugated anti-IgM, and PE-conjugated anti-CD43 antibodies. Pro–/pre–B cells were identified as B220 low IgM − , immature B cells were identified as B220 low IgM + , and mature B cells were identified as B220 high IgM + . Pro–B cells were identified as B220 low IgM − CD43 + and pre–B cells were identified as B220 low IgM − CD43 − . Results are shown as density plots (A and C) and as the absolute number of cells counted per femur (B and D). The percentage of cells in each gate is indicated on the plots. Bars represent mean values of pooled data from two experiments, and the closed circles are individual mice. *, P
Figure Legend Snippet: Reduced numbers of mature and immature B cells in the blood of B- S1pr1 KO mice. (A–D) Bone marrow B cell subpopulations from control and B- S1pr1 KO mice were analyzed by flow cytometry using FITC-conjugated anti-B220, APC-conjugated anti-IgM, and PE-conjugated anti-CD43 antibodies. Pro–/pre–B cells were identified as B220 low IgM − , immature B cells were identified as B220 low IgM + , and mature B cells were identified as B220 high IgM + . Pro–B cells were identified as B220 low IgM − CD43 + and pre–B cells were identified as B220 low IgM − CD43 − . Results are shown as density plots (A and C) and as the absolute number of cells counted per femur (B and D). The percentage of cells in each gate is indicated on the plots. Bars represent mean values of pooled data from two experiments, and the closed circles are individual mice. *, P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

Immature B cells in the bone marrow do not efficiently enter blood and have elevated apoptosis in the absence of S1P1 receptor. (A–D) Mice were pulsed with BrdU, and B cells from the bone marrow (A and B) and peripheral blood (C and D) of control and B- S1pr1 KO mice were analyzed by flow cytometry using anti-B220, anti-IgD, and anti-IgM antibodies in combination with BrdU detection methodology, as described in Materials and methods. Results are shown as dot plots (A and C), and as absolute numbers of BrdU + B220 low IgM − (pro–/pre–) and B220 low IgM + (immature) B cells per femur (B) and BrdU + B220 + IgD low IgM high (immature) and B220 + IgD high IgM low (mature) B cells per 400 µl of blood (D). The percentage of cells in each gate is indicated on the plots. Bars represent mean values, and the closed circles are individual mice. Data are representative of three experiments with three to five mice of each genotype per experiment. *, P
Figure Legend Snippet: Immature B cells in the bone marrow do not efficiently enter blood and have elevated apoptosis in the absence of S1P1 receptor. (A–D) Mice were pulsed with BrdU, and B cells from the bone marrow (A and B) and peripheral blood (C and D) of control and B- S1pr1 KO mice were analyzed by flow cytometry using anti-B220, anti-IgD, and anti-IgM antibodies in combination with BrdU detection methodology, as described in Materials and methods. Results are shown as dot plots (A and C), and as absolute numbers of BrdU + B220 low IgM − (pro–/pre–) and B220 low IgM + (immature) B cells per femur (B) and BrdU + B220 + IgD low IgM high (immature) and B220 + IgD high IgM low (mature) B cells per 400 µl of blood (D). The percentage of cells in each gate is indicated on the plots. Bars represent mean values, and the closed circles are individual mice. Data are representative of three experiments with three to five mice of each genotype per experiment. *, P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

CD69 expression in bone marrow B cells modulates the appearance of immature B cells in peripheral blood. (A) CD69 expression on bone marrow B cells. Results are shown as anti-CD69 fluorescence intensity for cells from control and B- S1pr1 KO mice compared with the isotype control. Representative results are from nine independent experiments. (B) Expression of the human CD69 transgene on bone marrow total B cells in control and transgenic (CD69-Tg) mice. B220 + bone marrow cells were analyzed for their expression of human CD69 by flow cytometry. (C and D) Distribution of B220 low IgM − (pro–/pre–) and B220 low IgM + (immature) B cells in the bone marrow (C) and of B220 + IgD low IgM + (immature) and B220 + IgD high IgM low (mature) B cells in the peripheral blood (D) of control and CD69 transgenic mice. Bars represent mean values, and the closed circles are individual mice. Data represent pooled results from three experiments. **, P
Figure Legend Snippet: CD69 expression in bone marrow B cells modulates the appearance of immature B cells in peripheral blood. (A) CD69 expression on bone marrow B cells. Results are shown as anti-CD69 fluorescence intensity for cells from control and B- S1pr1 KO mice compared with the isotype control. Representative results are from nine independent experiments. (B) Expression of the human CD69 transgene on bone marrow total B cells in control and transgenic (CD69-Tg) mice. B220 + bone marrow cells were analyzed for their expression of human CD69 by flow cytometry. (C and D) Distribution of B220 low IgM − (pro–/pre–) and B220 low IgM + (immature) B cells in the bone marrow (C) and of B220 + IgD low IgM + (immature) and B220 + IgD high IgM low (mature) B cells in the peripheral blood (D) of control and CD69 transgenic mice. Bars represent mean values, and the closed circles are individual mice. Data represent pooled results from three experiments. **, P

Techniques Used: Expressing, Fluorescence, Mouse Assay, Transgenic Assay, Flow Cytometry, Cytometry

17) Product Images from "Developmental exposure to chemicals associated with unconventional oil and gas extraction alters immune homeostasis and viral immunity of the amphibian Xenopus"

Article Title: Developmental exposure to chemicals associated with unconventional oil and gas extraction alters immune homeostasis and viral immunity of the amphibian Xenopus

Journal: The Science of the total environment

doi: 10.1016/j.scitotenv.2019.03.395

Flow cytometry gating strategy (Flowjo 7). (A) Representative FACS bit map from splenocytes of a single animal: Y-axis (SSC-A): side light scatter (cell granularity and density). X-axis (FSC-A): light scatter (cell size). Two major cell populations were gated: (1) Larger cells (M9, DCs, PMN). (2) Smaller lymphocytes (B and T cells). Splenocytes were stained with mAbs specific for (C) MHC class II; (E) IgM/class II; (F) CD5/CD8 to identify CD8 and CD4 T cells (CD5+/CD8neg; (G) NK cells mAb. A negative control (B) was included for each staining.
Figure Legend Snippet: Flow cytometry gating strategy (Flowjo 7). (A) Representative FACS bit map from splenocytes of a single animal: Y-axis (SSC-A): side light scatter (cell granularity and density). X-axis (FSC-A): light scatter (cell size). Two major cell populations were gated: (1) Larger cells (M9, DCs, PMN). (2) Smaller lymphocytes (B and T cells). Splenocytes were stained with mAbs specific for (C) MHC class II; (E) IgM/class II; (F) CD5/CD8 to identify CD8 and CD4 T cells (CD5+/CD8neg; (G) NK cells mAb. A negative control (B) was included for each staining.

Techniques Used: Flow Cytometry, FACS, Staining, Negative Control

Effects of developmental exposure to an equimass mixture of 23 UOG chemicals on B and NK cells at steady state (sham-infected) (A) and during viral infection (B). Frequency and relative number of splenic IgM+ B cells and NK cells determined by flow cytometry (gate 2) in frogs developmentally exposed to 0.2 % ethanol (Ctrl; black) or 0.1 (gray) and 1.0 μg/L (white) of UOGs then either infected with FV3 for 3 or 6 days, or sham-infected. Xenopus-specific mAb used are: AM20 (class II); 10A9 (IgM); 1F8 (NK). These data are pools of 2 independent experiments (3 animals per group). * P
Figure Legend Snippet: Effects of developmental exposure to an equimass mixture of 23 UOG chemicals on B and NK cells at steady state (sham-infected) (A) and during viral infection (B). Frequency and relative number of splenic IgM+ B cells and NK cells determined by flow cytometry (gate 2) in frogs developmentally exposed to 0.2 % ethanol (Ctrl; black) or 0.1 (gray) and 1.0 μg/L (white) of UOGs then either infected with FV3 for 3 or 6 days, or sham-infected. Xenopus-specific mAb used are: AM20 (class II); 10A9 (IgM); 1F8 (NK). These data are pools of 2 independent experiments (3 animals per group). * P

Techniques Used: Infection, Flow Cytometry

18) Product Images from "The anti-apoptotic activities of Rel and RelA required during B-cell maturation involve the regulation of Bcl-2 expression"

Article Title: The anti-apoptotic activities of Rel and RelA required during B-cell maturation involve the regulation of Bcl-2 expression

Journal: The EMBO Journal

doi: 10.1093/emboj/19.23.6351

Fig. 2. Mature B cells are absent in rag-1 –/– mice engrafted with c- rel –/– rela –/– fetal liver cells. Cell suspensions from spleens of rag-1 –/– mice engrafted with control or c- rel –/– rela –/– fetal liver cells were stained with monoclonal antibodies specific for IgM, IgD, B220, CD21, CD22 and CD23, and analyzed by flow cytometry. Owing to the virtual absence of lymph nodes (LN) in rag-1 –/– mice that only receive c- rel –/– rela –/– fetal liver cells, LN stains (B220 versus Ly5.2) were performed on rag-1 –/– mice co-reconstituted with wild-type Ly5.1 + and Ly5.2 + control or c- rel –/– rela –/– fetal liver cells. The dot blots are representative of results obtained from three separate experiments using four or more animals from each genotype. Organ cellularity and the distribution of the relevant B-cell populations are indicated.
Figure Legend Snippet: Fig. 2. Mature B cells are absent in rag-1 –/– mice engrafted with c- rel –/– rela –/– fetal liver cells. Cell suspensions from spleens of rag-1 –/– mice engrafted with control or c- rel –/– rela –/– fetal liver cells were stained with monoclonal antibodies specific for IgM, IgD, B220, CD21, CD22 and CD23, and analyzed by flow cytometry. Owing to the virtual absence of lymph nodes (LN) in rag-1 –/– mice that only receive c- rel –/– rela –/– fetal liver cells, LN stains (B220 versus Ly5.2) were performed on rag-1 –/– mice co-reconstituted with wild-type Ly5.1 + and Ly5.2 + control or c- rel –/– rela –/– fetal liver cells. The dot blots are representative of results obtained from three separate experiments using four or more animals from each genotype. Organ cellularity and the distribution of the relevant B-cell populations are indicated.

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

Fig. 5. ( A ) RT–PCR analysis of A1 , bcl-x L and bcl-2 expression in control and c- rel –/– rela –/– splenic B cells. Cell suspensions from spleens of rag-1 –/– mice engrafted with control (wild-type, c- rel –/– or rela –/– ) or c- rel –/– rela –/– fetal liver cells were stained with antibodies specific for IgM and IgD. Typical staining profiles for control (wild-type, c- rel –/– or rela –/– ) and c- rel –/– rela –/– B-cell populations are depicted as FACS dot blots. Boxed regions correspond to the sIgM hi IgD lo (R1), IgM hi IgD hi (R2) and IgM lo IgD hi (R3; control only) B-cell populations purified by flow cytometry. Total RNA isolated from FACS-purified wild-type (lanes 1, 5 and 9), c- rel –/– (lanes 2, 6 and 10), rela –/– (lanes 3, 7 and 11) and c- rel –/– rela –/– (lanes 4 and 8) IgM hi IgD lo (lanes 1–4), IgM hi IgD hi (lanes 5–8) and IgM lo IgD hi (lanes 9–11) splenic B cells was subjected to semi-quantitative RT–PCR using bcl-2 , A1 , bcl-x L and β-actin-specific primers. PCR products were fractionated on 1% agarose gels. These data are representative of three independent experiments. ( B ) Bcl-2 levels are lower than normal in c- rel –/– rela –/– sIgM + B cells. Four-color immunofluorescence staining was used to identify sIgM hi IgD lo and sIgM hi IgD hi B cells, and to determine the intracellular levels of Bcl-2. Fixed, permeabilized wild-type or c- rel –/– rela –/– splenic B cells were stained with Cy5-labeled anti-B220, Texas Red-labeled anti-IgD and biotinylated anti-IgM antibodies, and revealed with R-phycoerythrin–streptavidin. Histograms depict the labeling intensity of wild-type B cells stained with an isotype-matched control antibody (gray line), or anti-Bcl-2 (3F11) FITC antibody stains of wild-type (broken line) and c- rel –/– rela –/– (solid line) B cells. ( C ) Mature c- rel –/– rela –/– T cells express normal levels of Bcl-2. Four-color immunofluorescence staining was performed to determine intracellular levels of Bcl-2 in wild-type and c- rel –/– rela –/– CD4 + and CD8 + T cells. Fixed, permeabilized splenic T cells (identified by staining with Cy-5-labeled anti-Thy-1) are shown as FACS dot-blot profiles (left panels, x -axis, staining with Texas Red-labeled anti-CD8 + antibodies; y -axis, staining with biotinylated anti-CD4 + antibodies plus R-phycoerythrin–streptavidin). The boxed regions R1 and R2 represent the gates for the CD4 + and CD8 + cells, respectively. The panels represent fluorescence intensity histograms of cells stained with control antibody (gray line) or anti-Bcl-2 antibody staining of wild-type (broken line) or c- rel –/– rela –/– (solid line) cells.
Figure Legend Snippet: Fig. 5. ( A ) RT–PCR analysis of A1 , bcl-x L and bcl-2 expression in control and c- rel –/– rela –/– splenic B cells. Cell suspensions from spleens of rag-1 –/– mice engrafted with control (wild-type, c- rel –/– or rela –/– ) or c- rel –/– rela –/– fetal liver cells were stained with antibodies specific for IgM and IgD. Typical staining profiles for control (wild-type, c- rel –/– or rela –/– ) and c- rel –/– rela –/– B-cell populations are depicted as FACS dot blots. Boxed regions correspond to the sIgM hi IgD lo (R1), IgM hi IgD hi (R2) and IgM lo IgD hi (R3; control only) B-cell populations purified by flow cytometry. Total RNA isolated from FACS-purified wild-type (lanes 1, 5 and 9), c- rel –/– (lanes 2, 6 and 10), rela –/– (lanes 3, 7 and 11) and c- rel –/– rela –/– (lanes 4 and 8) IgM hi IgD lo (lanes 1–4), IgM hi IgD hi (lanes 5–8) and IgM lo IgD hi (lanes 9–11) splenic B cells was subjected to semi-quantitative RT–PCR using bcl-2 , A1 , bcl-x L and β-actin-specific primers. PCR products were fractionated on 1% agarose gels. These data are representative of three independent experiments. ( B ) Bcl-2 levels are lower than normal in c- rel –/– rela –/– sIgM + B cells. Four-color immunofluorescence staining was used to identify sIgM hi IgD lo and sIgM hi IgD hi B cells, and to determine the intracellular levels of Bcl-2. Fixed, permeabilized wild-type or c- rel –/– rela –/– splenic B cells were stained with Cy5-labeled anti-B220, Texas Red-labeled anti-IgD and biotinylated anti-IgM antibodies, and revealed with R-phycoerythrin–streptavidin. Histograms depict the labeling intensity of wild-type B cells stained with an isotype-matched control antibody (gray line), or anti-Bcl-2 (3F11) FITC antibody stains of wild-type (broken line) and c- rel –/– rela –/– (solid line) B cells. ( C ) Mature c- rel –/– rela –/– T cells express normal levels of Bcl-2. Four-color immunofluorescence staining was performed to determine intracellular levels of Bcl-2 in wild-type and c- rel –/– rela –/– CD4 + and CD8 + T cells. Fixed, permeabilized splenic T cells (identified by staining with Cy-5-labeled anti-Thy-1) are shown as FACS dot-blot profiles (left panels, x -axis, staining with Texas Red-labeled anti-CD8 + antibodies; y -axis, staining with biotinylated anti-CD4 + antibodies plus R-phycoerythrin–streptavidin). The boxed regions R1 and R2 represent the gates for the CD4 + and CD8 + cells, respectively. The panels represent fluorescence intensity histograms of cells stained with control antibody (gray line) or anti-Bcl-2 antibody staining of wild-type (broken line) or c- rel –/– rela –/– (solid line) cells.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Mouse Assay, Staining, FACS, Purification, Flow Cytometry, Cytometry, Isolation, Quantitative RT-PCR, Polymerase Chain Reaction, Immunofluorescence, Labeling, Dot Blot, Fluorescence

19) Product Images from "Impaired humoral immunity in X-linked lymphoproliferative disease is associated with defective IL-10 production by CD4+ T cells"

Article Title: Impaired humoral immunity in X-linked lymphoproliferative disease is associated with defective IL-10 production by CD4+ T cells

Journal:

doi: 10.1172/JCI200523139

B cells fail to undergo isotype switching in vivo in XLP patients. PBMCs from 18 healthy donors, 12 XLP patients, and 18 CVID patients were labeled with mAbs specific for CD20, CD27, and either IgM, IgG, IgA, or an isotype control Ab. ( A ) Expression of
Figure Legend Snippet: B cells fail to undergo isotype switching in vivo in XLP patients. PBMCs from 18 healthy donors, 12 XLP patients, and 18 CVID patients were labeled with mAbs specific for CD20, CD27, and either IgM, IgG, IgA, or an isotype control Ab. ( A ) Expression of

Techniques Used: In Vivo, Labeling, Expressing

20) Product Images from "Development of Immunoglobulin M Memory to Both a T-Cell-Independent and a T-Cell-Dependent Antigen following Infection with Vibrio cholerae O1 in Bangladesh ▿"

Article Title: Development of Immunoglobulin M Memory to Both a T-Cell-Independent and a T-Cell-Dependent Antigen following Infection with Vibrio cholerae O1 in Bangladesh ▿

Journal: Infection and Immunity

doi: 10.1128/IAI.00868-09

Antigen-specific circulating ASCs of IgA, IgG, and IgM isotypes in cholera patients 2, 7, and 30 days after onset of illness. *, P
Figure Legend Snippet: Antigen-specific circulating ASCs of IgA, IgG, and IgM isotypes in cholera patients 2, 7, and 30 days after onset of illness. *, P

Techniques Used:

IgA, IgG, and IgM serological responses to CTB and LPS antigens in cholera patients at near baseline (day 2 after onset of illness), acute stage (day 7), and convalescence (day 30). *, Significant elevation ( P
Figure Legend Snippet: IgA, IgG, and IgM serological responses to CTB and LPS antigens in cholera patients at near baseline (day 2 after onset of illness), acute stage (day 7), and convalescence (day 30). *, Significant elevation ( P

Techniques Used: CtB Assay

Antigen-specific B-cell memory responses of IgA, IgG, and IgM isotypes in cholera patients 2, 30, and 90 days after onset of illness, determined from ELISA measurements of lymphocyte culture supernatant specimens. *, P
Figure Legend Snippet: Antigen-specific B-cell memory responses of IgA, IgG, and IgM isotypes in cholera patients 2, 30, and 90 days after onset of illness, determined from ELISA measurements of lymphocyte culture supernatant specimens. *, P

Techniques Used: Enzyme-linked Immunosorbent Assay

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Concentration Assay:

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Flow Cytometry:

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Purification:

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Incubation:

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Article Snippet: .. Plasma, NA-17, or control IgM (C48-6, anti-KLH; BD Biosciences — Pharmingen) diluted in 1% BSA-PBS was incubated with apoptotic thymocytes for 30 minutes at 4°C. .. Cells were washed and incubated with FITC-labeled rat anti-mouse IgM (II/41) in 1% BSA-PBS for 30 minutes and then washed again.

Staining:

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Article Title: Heterotrimeric G‐protein subunit Gαi2 contributes to agonist‐sensitive apoptosis and degranulation in murine platelets. Heterotrimeric G‐protein subunit Gαi2 contributes to agonist‐sensitive apoptosis and degranulation in murine platelets
Article Snippet: .. Subsequently, platelets were stained for 30 min with polyclonal fluorescein isothiocyanate (FITC) conjugated goat anti‐mouse IgG and IgM specific antibody (BD Pharmingen, Hamburg, Germany) at a concentration of 1:50 in Tyrode buffer. ..

Article Title: Enhanced eryptosis contributes to anemia in lung cancer patients
Article Snippet: .. After two washing steps with 100 μl PBS-BSA, cells were stained for 30 min with polyclonal fluorescein-isothiocyanate (FITC)-conjugated goat anti-mouse IgG and IgM specific antibody (1:50 dilution; BD Pharmingen, Hamburg, Germany) in PBS-BSA. ..

Article Title: Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing
Article Snippet: .. Isotype-specific information was obtained by staining microarrays with a panel of anti-human IgG1, IgA, IgG3 and IgM detection antibodies (BD Biosciences and Invitrogen). .. Microarrays from WNV E-coated slides were probed with mouse anti-human Igκ and Igλ (BD Biosciences) detection antibodies.

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    Becton Dickinson anti non gal igm
    Group 2 and 3: Graft histology. Histology of B256 (group 2, animal no. 1) on POD15 revealed the TK graft was injured, but not rejected (A, B). Immunohistochemistry showed frequent glomerular anti-non-Gal <t>IgM</t> and scattered anti-non-Gal <t>IgG</t> in few glomeruli
    Anti Non Gal Igm, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson anti igm biotin
    p50-NFκB1 is a direct negative regulator of the Il-6 gene in Fo B cells. (A) Nuclear/cytoplasmic translocation of p50-NFκB1 and p65/RelA was examined by Amnis ImageStream. Images show p65/RelA or p50-NFκB1 combined with a nuclear dye in WT and Nfκb1 −/− Fo B cells (B220 + <t>CD21</t> int <t>IgM</t> lo ). (B) Relative colocalization of p65/RelA or p50-NFκB1 with the nuclear dye in Fo B cells. (C and D) ChIP was performed on purified Fo B cells from WT and Nfκb1 −/− mice with an anti-p50, anti-p65/RelA, or isotype control Ab. (C) Schematic representation of four putative p50-NFκB1–binding sites in the Il-6 promoter. (D) Enrichment of Il-6 κB regions 1–4 as measured by qPCR (mean ± SEM). Data are from two to four independent experiments ( n ≥ 4 mice/genotype).
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    Group 2 and 3: Graft histology. Histology of B256 (group 2, animal no. 1) on POD15 revealed the TK graft was injured, but not rejected (A, B). Immunohistochemistry showed frequent glomerular anti-non-Gal IgM and scattered anti-non-Gal IgG in few glomeruli

    Journal: Transplantation

    Article Title: First Experience With the Use of a Recombinant CD3 Immunotoxin as Induction Therapy in Pig-to-Primate Xenotransplantation: The Effect of T-Cell Depletion on Outcome

    doi: 10.1097/TP.0b013e31822b92a5

    Figure Lengend Snippet: Group 2 and 3: Graft histology. Histology of B256 (group 2, animal no. 1) on POD15 revealed the TK graft was injured, but not rejected (A, B). Immunohistochemistry showed frequent glomerular anti-non-Gal IgM and scattered anti-non-Gal IgG in few glomeruli

    Article Snippet: Anti-non Gal IgM and IgG antibody were evaluated by indirect flow-cytometry using a Becton-Dickinson FACScan (Sunnyvale, CA).

    Techniques: Immunohistochemistry

    CD20 mAb treatment impairs allograft-specific IgG generation, but accelerates minor histocompatibility-mismatched skin allograft rejection and alloAg-specific CD4 + T cell proliferation. ( A ) C57BL/6 mice were given control or CD20 mAb 7 d before receiving BALB/C skin allografts. The graph shows allograft survival following control (dashed line; n=12) and CD20 (solid line; n=7) mAb treatment. ( B ) Serum was collected from mice in (A) 14 d after grafting and analyzed for BALB/C-specific IgM (top panels) and IgG (bottom panels) by indirect immunofluorescence staining with flow cytometry analysis. Flow cytometry histograms (left panels) show representative IgM and IgG staining of BALB/C-derived AG8 cells with serum from control (dashed line) and CD20 (solid line) mAb-treated mice, and untransplanted naïve (shaded) mice. Values (right panels) represent the MFI of stained cells from individual mice. Horizontal bars indicate mean MFI values for the group. ( C ) C57BL/6 mice were given control or CD20 mAb 7 d before receiving skin allografts from act-mOVA transgenic mice. The graph shows allograft survival following control (dashed line; n=24) and CD20 (solid line; n=21) mAb treatment. ( D-E ) C57BL/6 mice were given control or CD20 mAb 6 d before receiving CFSE-labeled Thy1.1 + CD4 + T cells from OT-II mice. One d later, the mice received Act-mOVA skin grafts. (D) Draining LN and (E) spleen lymphocytes were isolated 5 d later, with CD4 expression and CFSE dilution of Thy1.1 + T cells assessed by immunofluorescence staining with flow cytometry analysis. Representative CFSE versus cell surface CD4 staining for Thy1.1 + cells is shown, with the percentages of CFSE-diluted CD4 + cells within each gate indicated as a fraction of total CD4 + Thy1.1 + T cells. Values (right panels) represent the percentage of CFSE-diluted cells from individual control mAb- and CD20 mAb-treated mice. Horizontal bars indicate mean values for groups. Significant differences between control mAb and CD20 mAb sample means are indicated: *, p

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

    Article Title: B Lymphocytes Differentially Influence Acute and Chronic Allograft Rejection in Mice 1

    doi: 10.4049/jimmunol.1002983

    Figure Lengend Snippet: CD20 mAb treatment impairs allograft-specific IgG generation, but accelerates minor histocompatibility-mismatched skin allograft rejection and alloAg-specific CD4 + T cell proliferation. ( A ) C57BL/6 mice were given control or CD20 mAb 7 d before receiving BALB/C skin allografts. The graph shows allograft survival following control (dashed line; n=12) and CD20 (solid line; n=7) mAb treatment. ( B ) Serum was collected from mice in (A) 14 d after grafting and analyzed for BALB/C-specific IgM (top panels) and IgG (bottom panels) by indirect immunofluorescence staining with flow cytometry analysis. Flow cytometry histograms (left panels) show representative IgM and IgG staining of BALB/C-derived AG8 cells with serum from control (dashed line) and CD20 (solid line) mAb-treated mice, and untransplanted naïve (shaded) mice. Values (right panels) represent the MFI of stained cells from individual mice. Horizontal bars indicate mean MFI values for the group. ( C ) C57BL/6 mice were given control or CD20 mAb 7 d before receiving skin allografts from act-mOVA transgenic mice. The graph shows allograft survival following control (dashed line; n=24) and CD20 (solid line; n=21) mAb treatment. ( D-E ) C57BL/6 mice were given control or CD20 mAb 6 d before receiving CFSE-labeled Thy1.1 + CD4 + T cells from OT-II mice. One d later, the mice received Act-mOVA skin grafts. (D) Draining LN and (E) spleen lymphocytes were isolated 5 d later, with CD4 expression and CFSE dilution of Thy1.1 + T cells assessed by immunofluorescence staining with flow cytometry analysis. Representative CFSE versus cell surface CD4 staining for Thy1.1 + cells is shown, with the percentages of CFSE-diluted CD4 + cells within each gate indicated as a fraction of total CD4 + Thy1.1 + T cells. Values (right panels) represent the percentage of CFSE-diluted cells from individual control mAb- and CD20 mAb-treated mice. Horizontal bars indicate mean values for groups. Significant differences between control mAb and CD20 mAb sample means are indicated: *, p

    Article Snippet: The cells were then washed three times, incubated with fluorochrome-conjugated secondary polyclonal anti-mouse IgG or IgM Abs, and analyzed by flow cytometry on a FACScan flow cytometer (BD).

    Techniques: Mouse Assay, Immunofluorescence, Staining, Flow Cytometry, Cytometry, Derivative Assay, Activated Clotting Time Assay, Transgenic Assay, Labeling, Isolation, Expressing

    CD19 mAb treatment depletes serum IgG, while CD20 mAb treatment does not. Two mo-old C57BL/6 huCD19Tg mice were given control, CD20, or CD19 mAb on day 0, with serum harvested at the indicated time points for analysis by ELISA. ( A-B ) Values represent serum IgM (A) and IgG (B) concentrations from individual mice receiving the indicated mAb. Solid lines connect mean serum Ig concentrations for each group. Significant differences between sample means relative to day 0 are indicated: **, p

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

    Article Title: B Lymphocytes Differentially Influence Acute and Chronic Allograft Rejection in Mice 1

    doi: 10.4049/jimmunol.1002983

    Figure Lengend Snippet: CD19 mAb treatment depletes serum IgG, while CD20 mAb treatment does not. Two mo-old C57BL/6 huCD19Tg mice were given control, CD20, or CD19 mAb on day 0, with serum harvested at the indicated time points for analysis by ELISA. ( A-B ) Values represent serum IgM (A) and IgG (B) concentrations from individual mice receiving the indicated mAb. Solid lines connect mean serum Ig concentrations for each group. Significant differences between sample means relative to day 0 are indicated: **, p

    Article Snippet: The cells were then washed three times, incubated with fluorochrome-conjugated secondary polyclonal anti-mouse IgG or IgM Abs, and analyzed by flow cytometry on a FACScan flow cytometer (BD).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    CD19 mAb treatment depletes Ab-secreting plasma cells. ( A ) CD19 mAb depletes both short-lived and long-lived CD138 hi plasma cells. C57BL/6–129 huCD19Tg mice were fed BrdU for 7 d prior to and following control, CD20, or CD19 mAb treatments, with BM and spleen cells analyzed for surface CD138 and intracellular BrdU staining on day 7. Dot plots show mean (± SEM) percentages of CD138 hi BrdU + (short-lived plasma cells) and CD138 hi BrdU − (long-lived plasma cells). Bar graphs indicate mean numbers of CD138 hi BrdU + and CD138 hi BrdU − cells from mice receiving the indicated treatment (n=8 mice per group). ( B ) C57BL/6 huCD19Tg mice were given control, CD20, or CD19 mAb, with BM and spleen Ab-secreting cell (ASC) numbers determined 7 d later by ELISPOT analysis. Bar graphs indicate mean (± SEM) numbers of IgM and IgG ASCs (n=4 mice per group). ( C ) CD19 expression is maintained on CD138 + plasmablasts, while CD20 expression decreases. BM and spleen cells from naive C57BL/6–129 huCD19Tg mice were co-stained for CD138, B220, CD20, and CD19. Representative dot plots (left panels) indicate the B220CD138 − , B220 + CD138 − , B220 + CD138 hi , and B220 lo CD138 hi cell populations that were analyzed for CD20 and CD19 expression (right panels). Vertical dashed lines indicate the MFI of CD20 or CD19 expression by splenic B220 + CD138 − B cells. Bar graphs indicate mean (± SEM) levels of CD20 or CD19 expression relative to spleen B220 + CD138 − B cells. Background staining MFI values (

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

    Article Title: B Lymphocytes Differentially Influence Acute and Chronic Allograft Rejection in Mice 1

    doi: 10.4049/jimmunol.1002983

    Figure Lengend Snippet: CD19 mAb treatment depletes Ab-secreting plasma cells. ( A ) CD19 mAb depletes both short-lived and long-lived CD138 hi plasma cells. C57BL/6–129 huCD19Tg mice were fed BrdU for 7 d prior to and following control, CD20, or CD19 mAb treatments, with BM and spleen cells analyzed for surface CD138 and intracellular BrdU staining on day 7. Dot plots show mean (± SEM) percentages of CD138 hi BrdU + (short-lived plasma cells) and CD138 hi BrdU − (long-lived plasma cells). Bar graphs indicate mean numbers of CD138 hi BrdU + and CD138 hi BrdU − cells from mice receiving the indicated treatment (n=8 mice per group). ( B ) C57BL/6 huCD19Tg mice were given control, CD20, or CD19 mAb, with BM and spleen Ab-secreting cell (ASC) numbers determined 7 d later by ELISPOT analysis. Bar graphs indicate mean (± SEM) numbers of IgM and IgG ASCs (n=4 mice per group). ( C ) CD19 expression is maintained on CD138 + plasmablasts, while CD20 expression decreases. BM and spleen cells from naive C57BL/6–129 huCD19Tg mice were co-stained for CD138, B220, CD20, and CD19. Representative dot plots (left panels) indicate the B220CD138 − , B220 + CD138 − , B220 + CD138 hi , and B220 lo CD138 hi cell populations that were analyzed for CD20 and CD19 expression (right panels). Vertical dashed lines indicate the MFI of CD20 or CD19 expression by splenic B220 + CD138 − B cells. Bar graphs indicate mean (± SEM) levels of CD20 or CD19 expression relative to spleen B220 + CD138 − B cells. Background staining MFI values (

    Article Snippet: The cells were then washed three times, incubated with fluorochrome-conjugated secondary polyclonal anti-mouse IgG or IgM Abs, and analyzed by flow cytometry on a FACScan flow cytometer (BD).

    Techniques: Mouse Assay, BrdU Staining, Enzyme-linked Immunospot, Expressing, Staining

    CD19 mAb treatment impairs allograft-specific IgG production. C57BL/6–129 huCD19Tg mice were given control, CD20, or CD19 mAb 7 d before receiving a fully MHC-mismatched DBA/2 heterotopic cardiac allograft. ( A ) Allograft survival following control (n=5, dashed line), CD20 (n=4, solid line), or CD19 (n=4, heavy solid line) mAb treatment. ( B ) Serum was collected from mice in (A) 14 d after grafting and analyzed for DBA/2-specific IgM (top panels) and IgG (bottom panels) by indirect immunofluorescence staining with flow cytometry analysis. Flow cytometry histograms (left panels) show representative IgM and IgG staining of DBA/2-derived P815 cells with serum from control (dashed line), CD20 (solid line), and CD19 (heavy solid line) mAb-treated mice, and untransplanted naïve mice (shaded). Values (right panels) represent the MFI of stained cells from individual mice that received the indicated treatment. Horizontal bars indicate mean MFI values for each group. Significant differences between control and CD20 or CD19 mAb sample means, or between specified means, are indicated: *, p

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

    Article Title: B Lymphocytes Differentially Influence Acute and Chronic Allograft Rejection in Mice 1

    doi: 10.4049/jimmunol.1002983

    Figure Lengend Snippet: CD19 mAb treatment impairs allograft-specific IgG production. C57BL/6–129 huCD19Tg mice were given control, CD20, or CD19 mAb 7 d before receiving a fully MHC-mismatched DBA/2 heterotopic cardiac allograft. ( A ) Allograft survival following control (n=5, dashed line), CD20 (n=4, solid line), or CD19 (n=4, heavy solid line) mAb treatment. ( B ) Serum was collected from mice in (A) 14 d after grafting and analyzed for DBA/2-specific IgM (top panels) and IgG (bottom panels) by indirect immunofluorescence staining with flow cytometry analysis. Flow cytometry histograms (left panels) show representative IgM and IgG staining of DBA/2-derived P815 cells with serum from control (dashed line), CD20 (solid line), and CD19 (heavy solid line) mAb-treated mice, and untransplanted naïve mice (shaded). Values (right panels) represent the MFI of stained cells from individual mice that received the indicated treatment. Horizontal bars indicate mean MFI values for each group. Significant differences between control and CD20 or CD19 mAb sample means, or between specified means, are indicated: *, p

    Article Snippet: The cells were then washed three times, incubated with fluorochrome-conjugated secondary polyclonal anti-mouse IgG or IgM Abs, and analyzed by flow cytometry on a FACScan flow cytometer (BD).

    Techniques: Mouse Assay, Immunofluorescence, Staining, Flow Cytometry, Cytometry, Derivative Assay

    CD19 mAb treatment prevents renal allograft rejection, impairs allograft-specific IgG generation, and reduces pre-existing serum IgG levels. C57BL/6–129 huCD19Tg mice were given control, CD20, or CD19 mAb 7 d before receiving a DBA/2 renal allograft. A nephrectomy was performed on the remaining kidney 1–3 d following allografting, and mouse survival was monitored daily as a measure of allograft rejection. ( A ) Allograft survival following control (n=9, dashed line), CD20 (n=8, solid line), or CD19 (n=7, heavy solid line) mAb treatments. ( B ) Serum was collected from mice 21 d after grafting and analyzed for DBA/2-specific IgM (top panels) and IgG (bottom panels) by indirect immunofluorescence staining with flow cytometry analysis. Flow cytometry histograms (left panels) show representative IgM and IgG staining of P815 cells with serum from control (dashed line), CD20 (solid line), and CD19 (heavy solid line) mAb-treated mice, and untransplanted naïve (shaded) mice. Values (right panels) represent the results from individual mice that received the indicated treatment. Horizontal bars indicate mean MFI values for groups. ( C ) C57BL/6–129 huCD19Tg mice were immunized with DBA/2 splenocytes at week 0 and boosted at week 2. Mice were then randomized and given control, CD20, or CD19 mAb at week 5. Serum was harvested at the indicated time points, with DBA/2-specific IgG measured by indirect immunofluorescence staining with flow cytometry analysis. Values represent the mean percent change in MFI relative to week 5 (n=4–5 mice per group). Significant differences between control and CD20 or CD19 mAb sample means, or between specified means, are indicated: *, p

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

    Article Title: B Lymphocytes Differentially Influence Acute and Chronic Allograft Rejection in Mice 1

    doi: 10.4049/jimmunol.1002983

    Figure Lengend Snippet: CD19 mAb treatment prevents renal allograft rejection, impairs allograft-specific IgG generation, and reduces pre-existing serum IgG levels. C57BL/6–129 huCD19Tg mice were given control, CD20, or CD19 mAb 7 d before receiving a DBA/2 renal allograft. A nephrectomy was performed on the remaining kidney 1–3 d following allografting, and mouse survival was monitored daily as a measure of allograft rejection. ( A ) Allograft survival following control (n=9, dashed line), CD20 (n=8, solid line), or CD19 (n=7, heavy solid line) mAb treatments. ( B ) Serum was collected from mice 21 d after grafting and analyzed for DBA/2-specific IgM (top panels) and IgG (bottom panels) by indirect immunofluorescence staining with flow cytometry analysis. Flow cytometry histograms (left panels) show representative IgM and IgG staining of P815 cells with serum from control (dashed line), CD20 (solid line), and CD19 (heavy solid line) mAb-treated mice, and untransplanted naïve (shaded) mice. Values (right panels) represent the results from individual mice that received the indicated treatment. Horizontal bars indicate mean MFI values for groups. ( C ) C57BL/6–129 huCD19Tg mice were immunized with DBA/2 splenocytes at week 0 and boosted at week 2. Mice were then randomized and given control, CD20, or CD19 mAb at week 5. Serum was harvested at the indicated time points, with DBA/2-specific IgG measured by indirect immunofluorescence staining with flow cytometry analysis. Values represent the mean percent change in MFI relative to week 5 (n=4–5 mice per group). Significant differences between control and CD20 or CD19 mAb sample means, or between specified means, are indicated: *, p

    Article Snippet: The cells were then washed three times, incubated with fluorochrome-conjugated secondary polyclonal anti-mouse IgG or IgM Abs, and analyzed by flow cytometry on a FACScan flow cytometer (BD).

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

    Immunoglobulin expression is needed for ER maintenance in resting B cells. (A) Mature resting B cells from B1-8 flox (B18f) and B1-8 flox × R26Stop flox P110* (B18f P110*) mice were treated with TAT-Cre to induce the deletion of loxP-flanked sequences. The frequency of BCR-negative B cells was monitored over time. (B) Cell size of B1-8 flox × Mb1-creERT2 B cells treated with tamoxifen to induce the deletion of the Ig heavy chain. (B, C) Cell size (B) and ER mass (C) of cells positive for surface IgM (IgM+) and negative for surface IgM (IgM−) was determined on day 3. Data were tested for significance using the paired t test. (A, B) N = 3; P = 0.75 (A), ** P = 0.0087 (B). Experiments were performed in six technical replicates. Circles indicate mean values of these replicates obtained from three different mice. (D) Mature resting B cells from B1-8 flox × EµBcl2 mice treated with TAT-Cre to induce the deletion of the Ig heavy chain, FACS-sorted on d5 upon TAT-Cre, and let to recover for 24 h post-sorting were resuspended in medium containing pyruvate, glutamine, and glucose, and OCR was measured using Seahorse flux technology on d6. The measurements were performed in technical triplicates and are displayed as mean ± SD. a, oligomycin; b, FCCP; c, rotenone + antimycin. (E) mRNA levels for the indicated genes were determined on day 2 after TAT-Cre–mediated deletion of loxP-flanked sequences in B18f and B18f P110* B cells positive or negative for surface IgM expression. Results were tested for significance using two-way ANOVA. For clarity, only differences between IgM+/IgM− and IgM+ P110*/IgM− P110* cells are shown. ** P = 0.0078 and 0.0057, * P = 0.0351 and 0.0402. Graph shows data obtained from three mice. (F, G, H) B cells from B1-8 flox × EµBcl2 mice were purified, treated with TAT-Cre, kept for 4–6 d in culture to allow for IgM deletion, and stimulated overnight with LPS. (F, G, H) BiP protein levels (F), cell size (G), and CD69 surface levels (H) were assessed by flow cytometry. Results were tested for significance using ANOVA. For clarity only differences between IgM+ and IgM− cells are shown. Circles represent data obtained from different mice. If samples were measured in technical replicates, circles represent mean values obtained from these replicates. For F: N = 7, ** P = 0.002; for G: N = 4, *** P = 0.0006; for H: N = 5, * P = 0.0192. Heavy chain deletion was performed in an in vitro cell culture in the presence of the cytokine BAFF.

    Journal: Life Science Alliance

    Article Title: Immunoglobulin expression in the endoplasmic reticulum shapes the metabolic fitness of B lymphocytes

    doi: 10.26508/lsa.202000700

    Figure Lengend Snippet: Immunoglobulin expression is needed for ER maintenance in resting B cells. (A) Mature resting B cells from B1-8 flox (B18f) and B1-8 flox × R26Stop flox P110* (B18f P110*) mice were treated with TAT-Cre to induce the deletion of loxP-flanked sequences. The frequency of BCR-negative B cells was monitored over time. (B) Cell size of B1-8 flox × Mb1-creERT2 B cells treated with tamoxifen to induce the deletion of the Ig heavy chain. (B, C) Cell size (B) and ER mass (C) of cells positive for surface IgM (IgM+) and negative for surface IgM (IgM−) was determined on day 3. Data were tested for significance using the paired t test. (A, B) N = 3; P = 0.75 (A), ** P = 0.0087 (B). Experiments were performed in six technical replicates. Circles indicate mean values of these replicates obtained from three different mice. (D) Mature resting B cells from B1-8 flox × EµBcl2 mice treated with TAT-Cre to induce the deletion of the Ig heavy chain, FACS-sorted on d5 upon TAT-Cre, and let to recover for 24 h post-sorting were resuspended in medium containing pyruvate, glutamine, and glucose, and OCR was measured using Seahorse flux technology on d6. The measurements were performed in technical triplicates and are displayed as mean ± SD. a, oligomycin; b, FCCP; c, rotenone + antimycin. (E) mRNA levels for the indicated genes were determined on day 2 after TAT-Cre–mediated deletion of loxP-flanked sequences in B18f and B18f P110* B cells positive or negative for surface IgM expression. Results were tested for significance using two-way ANOVA. For clarity, only differences between IgM+/IgM− and IgM+ P110*/IgM− P110* cells are shown. ** P = 0.0078 and 0.0057, * P = 0.0351 and 0.0402. Graph shows data obtained from three mice. (F, G, H) B cells from B1-8 flox × EµBcl2 mice were purified, treated with TAT-Cre, kept for 4–6 d in culture to allow for IgM deletion, and stimulated overnight with LPS. (F, G, H) BiP protein levels (F), cell size (G), and CD69 surface levels (H) were assessed by flow cytometry. Results were tested for significance using ANOVA. For clarity only differences between IgM+ and IgM− cells are shown. Circles represent data obtained from different mice. If samples were measured in technical replicates, circles represent mean values obtained from these replicates. For F: N = 7, ** P = 0.002; for G: N = 4, *** P = 0.0006; for H: N = 5, * P = 0.0192. Heavy chain deletion was performed in an in vitro cell culture in the presence of the cytokine BAFF.

    Article Snippet: Primary mouse B cells were stained with anti-B220 (RA3-6B2), anti-CD19 (6D5), anti-IgM (II/41), and anti-CD69 (H1.2F3), and for cell sorting on Aria (BD), Fab fragment anti-IgM antibody (Jackson) was used.

    Techniques: Expressing, Mouse Assay, FACS, Purification, Flow Cytometry, In Vitro, Cell Culture

    BCR expression boosts B lymphoma fitness but is not absolutely required for survival. (A) Cells were stained with anti-IgM and anti-IgD to examine cell surface expression of the BCR. Anti-CD19 was used as a control. (B) Expression of the μ-heavy chain, λ-light chain, Igα, and Igβ were determined by Western blot. One of three independent experiments is shown. (C) The cells were plated on d0 and cell numbers were assessed on d1, d2, and d3 using the CCK8-kit. Values were normalized to the measurement obtained on d1. Significance was determined using the ANOVA test. (D) Expression of the indicated proteins was determined by Western blot. One of four independent experiments is shown. (E) Forward scatter (FSC-A) as a measure of cell size was determined using flow cytometry. Significance was determined using the Mann–Whitney test. N = 8. (F) Biological triplicates of Ig heavy chain–deficient DG75 cells were sorted on d3 upon Ig heavy chain deletion and pooled for analysis. Expression of the Ig heavy chain (antibody directed against the CH domain) and histone 3 (H3) was determined by Western blot. One of two independent experiments is shown. (G) Cell size measured on d6 after Ig heavy chain deletion. Significance was determined using the paired t test. N = 3; ** P = 0.0074. (H) Relative abundance of WT and H-KO DG75 cells in a mixed culture at the indicated time points after Ig heavy chain deletion. Linear regression analysis was performed. Slopes of the WT and KO abundance lines were found to be significantly different. P

    Journal: Life Science Alliance

    Article Title: Immunoglobulin expression in the endoplasmic reticulum shapes the metabolic fitness of B lymphocytes

    doi: 10.26508/lsa.202000700

    Figure Lengend Snippet: BCR expression boosts B lymphoma fitness but is not absolutely required for survival. (A) Cells were stained with anti-IgM and anti-IgD to examine cell surface expression of the BCR. Anti-CD19 was used as a control. (B) Expression of the μ-heavy chain, λ-light chain, Igα, and Igβ were determined by Western blot. One of three independent experiments is shown. (C) The cells were plated on d0 and cell numbers were assessed on d1, d2, and d3 using the CCK8-kit. Values were normalized to the measurement obtained on d1. Significance was determined using the ANOVA test. (D) Expression of the indicated proteins was determined by Western blot. One of four independent experiments is shown. (E) Forward scatter (FSC-A) as a measure of cell size was determined using flow cytometry. Significance was determined using the Mann–Whitney test. N = 8. (F) Biological triplicates of Ig heavy chain–deficient DG75 cells were sorted on d3 upon Ig heavy chain deletion and pooled for analysis. Expression of the Ig heavy chain (antibody directed against the CH domain) and histone 3 (H3) was determined by Western blot. One of two independent experiments is shown. (G) Cell size measured on d6 after Ig heavy chain deletion. Significance was determined using the paired t test. N = 3; ** P = 0.0074. (H) Relative abundance of WT and H-KO DG75 cells in a mixed culture at the indicated time points after Ig heavy chain deletion. Linear regression analysis was performed. Slopes of the WT and KO abundance lines were found to be significantly different. P

    Article Snippet: Primary mouse B cells were stained with anti-B220 (RA3-6B2), anti-CD19 (6D5), anti-IgM (II/41), and anti-CD69 (H1.2F3), and for cell sorting on Aria (BD), Fab fragment anti-IgM antibody (Jackson) was used.

    Techniques: Expressing, Staining, Western Blot, Flow Cytometry, MANN-WHITNEY

    p50-NFκB1 is a direct negative regulator of the Il-6 gene in Fo B cells. (A) Nuclear/cytoplasmic translocation of p50-NFκB1 and p65/RelA was examined by Amnis ImageStream. Images show p65/RelA or p50-NFκB1 combined with a nuclear dye in WT and Nfκb1 −/− Fo B cells (B220 + CD21 int IgM lo ). (B) Relative colocalization of p65/RelA or p50-NFκB1 with the nuclear dye in Fo B cells. (C and D) ChIP was performed on purified Fo B cells from WT and Nfκb1 −/− mice with an anti-p50, anti-p65/RelA, or isotype control Ab. (C) Schematic representation of four putative p50-NFκB1–binding sites in the Il-6 promoter. (D) Enrichment of Il-6 κB regions 1–4 as measured by qPCR (mean ± SEM). Data are from two to four independent experiments ( n ≥ 4 mice/genotype).

    Journal: The Journal of Experimental Medicine

    Article Title: NFκB1 is essential to prevent the development of multiorgan autoimmunity by limiting IL-6 production in follicular B cells

    doi: 10.1084/jem.20151182

    Figure Lengend Snippet: p50-NFκB1 is a direct negative regulator of the Il-6 gene in Fo B cells. (A) Nuclear/cytoplasmic translocation of p50-NFκB1 and p65/RelA was examined by Amnis ImageStream. Images show p65/RelA or p50-NFκB1 combined with a nuclear dye in WT and Nfκb1 −/− Fo B cells (B220 + CD21 int IgM lo ). (B) Relative colocalization of p65/RelA or p50-NFκB1 with the nuclear dye in Fo B cells. (C and D) ChIP was performed on purified Fo B cells from WT and Nfκb1 −/− mice with an anti-p50, anti-p65/RelA, or isotype control Ab. (C) Schematic representation of four putative p50-NFκB1–binding sites in the Il-6 promoter. (D) Enrichment of Il-6 κB regions 1–4 as measured by qPCR (mean ± SEM). Data are from two to four independent experiments ( n ≥ 4 mice/genotype).

    Article Snippet: Abs and flow cytometry Surface staining for flow cytometric analysis was performed using the following fluorochrome-conjugated mAbs: anti–CD19-FITC (1D3), anti–CD24-FITC (M1/69), anti–B220-APCCy7 (RA3-6B2), anti–PD-1–PE (J43), anti–CXCR5-APC (2G8), anti–CD21-APC (7G6), anti–CD45.2-biotin (104), anti–ICOS-biotin (7E.17G9), anti–IgM-biotin (R6-60.2), anti–CD86-FITC (GL1), anti–CD80-PE (16-10A1), anti–MHC class II–biotin (AF6-120.1), anti–IgG1-biotin (A85-1), anti–FAS-biotin (Jo2), anti–CD8-APCCy7 (53-6.7), anti–CD11b-APC (M1/70), and anti–CD3-PE (145-2C11; all from BD); and anti–CD23-FITC, -APC (B3B4), anti-CD4-PE, -biotin (GK1.5), anti–CD45.1-APC, -FITC (A20), anti–CD40-biotin (1C10), and anti–GL7-eFluor660 (GL-7; all from eBioscience).

    Techniques: Translocation Assay, Chromatin Immunoprecipitation, Purification, Mouse Assay, Binding Assay, Real-time Polymerase Chain Reaction

    Enhanced B cell differentiation is the result of the cell-intrinsic loss of NFκB1. (A–F) mBM chimera mice were generated by mixing ly5.1 + WT BM cells with Nfκb1 −/− or WT (both ly5.2 + ) BM cells (1:1 ratio) and transferred into lethally irradiated ly5.1 + WT hosts. After 16 wk, spleens were isolated and processed for flow cytometry analysis. Splenocytes were defined on the basis of ly5.2 expression. (A) Ki67 expression in Fo B cells (B220 + CD21 int IgM lo ); dot plots show GL7 versus Bcl6 staining gated on B220 + cells (GCs; GL7 + Bcl6 + ) and IgG1 staining versus FSC gated on Fo B cells (IgG1 + B cells). The numbers represent percentages. (B) Absolute cell numbers of Ki67 + Fo B cells, GC B cells, and IgG1 + B cells. (C) Expression of Bcl-6 in B cell subsets derived from WT or Nfκb1 −/− BM cells in mBM chimeric mice. (D) Bcl-6 expression (mean fluorescence intensity [MFI]) in Fo and GC B cells. (E) Absolute numbers of WT and Nfκb1 −/− T FH cells (CD4 + CXCR5 + PD-1 + ). (F) Intracellular IL-6 expression in Fo B cells from mBM chimera mice. Results were derived from two independent cohorts of reconstituted mice ( n = 6/group). Statistical significance was determined using independent or paired samples Student's t tests as appropriate. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: NFκB1 is essential to prevent the development of multiorgan autoimmunity by limiting IL-6 production in follicular B cells

    doi: 10.1084/jem.20151182

    Figure Lengend Snippet: Enhanced B cell differentiation is the result of the cell-intrinsic loss of NFκB1. (A–F) mBM chimera mice were generated by mixing ly5.1 + WT BM cells with Nfκb1 −/− or WT (both ly5.2 + ) BM cells (1:1 ratio) and transferred into lethally irradiated ly5.1 + WT hosts. After 16 wk, spleens were isolated and processed for flow cytometry analysis. Splenocytes were defined on the basis of ly5.2 expression. (A) Ki67 expression in Fo B cells (B220 + CD21 int IgM lo ); dot plots show GL7 versus Bcl6 staining gated on B220 + cells (GCs; GL7 + Bcl6 + ) and IgG1 staining versus FSC gated on Fo B cells (IgG1 + B cells). The numbers represent percentages. (B) Absolute cell numbers of Ki67 + Fo B cells, GC B cells, and IgG1 + B cells. (C) Expression of Bcl-6 in B cell subsets derived from WT or Nfκb1 −/− BM cells in mBM chimeric mice. (D) Bcl-6 expression (mean fluorescence intensity [MFI]) in Fo and GC B cells. (E) Absolute numbers of WT and Nfκb1 −/− T FH cells (CD4 + CXCR5 + PD-1 + ). (F) Intracellular IL-6 expression in Fo B cells from mBM chimera mice. Results were derived from two independent cohorts of reconstituted mice ( n = 6/group). Statistical significance was determined using independent or paired samples Student's t tests as appropriate. *, P

    Article Snippet: Abs and flow cytometry Surface staining for flow cytometric analysis was performed using the following fluorochrome-conjugated mAbs: anti–CD19-FITC (1D3), anti–CD24-FITC (M1/69), anti–B220-APCCy7 (RA3-6B2), anti–PD-1–PE (J43), anti–CXCR5-APC (2G8), anti–CD21-APC (7G6), anti–CD45.2-biotin (104), anti–ICOS-biotin (7E.17G9), anti–IgM-biotin (R6-60.2), anti–CD86-FITC (GL1), anti–CD80-PE (16-10A1), anti–MHC class II–biotin (AF6-120.1), anti–IgG1-biotin (A85-1), anti–FAS-biotin (Jo2), anti–CD8-APCCy7 (53-6.7), anti–CD11b-APC (M1/70), and anti–CD3-PE (145-2C11; all from BD); and anti–CD23-FITC, -APC (B3B4), anti-CD4-PE, -biotin (GK1.5), anti–CD45.1-APC, -FITC (A20), anti–CD40-biotin (1C10), and anti–GL7-eFluor660 (GL-7; all from eBioscience).

    Techniques: Cell Differentiation, Mouse Assay, Generated, Irradiation, Isolation, Flow Cytometry, Cytometry, Expressing, Staining, Derivative Assay, Fluorescence