Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A and B ) Naive B cells purified from the spleen of Igk −/− B1-8 flox/+ mice were stimulated with NP-CGG or NP-Ficoll. ( A ) 3 H-thymidine incorporation on day 3. ( B ) IgM and IgG concentrations in the culture supernatants on day 3. ( C–E ) Igk −/− B1-8 flox/+ B cells were stimulated with none (–), NP-CGG, or NP-Ficoll and IL-1α, IL-1β, or IFNα, as indicated. ( C ) Enzyme-linked immunosorbent assay (ELISA) of IgG3 in the culture supernatant on day 3. AU, arbitrary units. ( D ) Representative flow cytometric plots on day 3 with the numbers indicating percentages of IgG3 + B cells (top). The frequencies of IgG3 + cells among the B cells (bottom). ( E ) quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of the circle Iγ3-Cµ and Aicda transcripts on day 2. Data are means ± standard deviations (SDs) of two ( B and C ), two to three ( D ), or three ( A ) biological replicates or three technical replicates ( E ). The data are representative of at least three ( A and B ) or two ( C–E ) independent experiments. *p < 0.05; ***p < 0.001; ****p < 0.0001; p values were calculated by one- ( B ) or two ( C and D )-way analysis of variance (ANOVA) with Tukey’s test. Figure 1—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A–C ) Igk −/− B1-8 flox/+ naive B cells were stimulated with nothing (–), NP-CGG, or NP-Ficoll in the presence of LPS, R-848, or CpG. ( A ) IgG concentration in the culture supernatants on day 3. ( B ) Representative flow cytometric plots on day 3 showing percentages of IgG + B cells. ( C ) qRT-PCR analysis of Aicda transcripts on day 2. ( D and E ) Igk −/− B1-8 flox/+ naive B cells were stimulated with NP-Ficoll and each indicated cytokine. ( D ) IgG3 in the culture supernatants on day 4 titrated by enzyme-linked immunosorbent assay (ELISA). AU, arbitrary units. ( E ) Representative flow cytometric profiles of IgG1, IgG2b, IgG2c, and IgG3 on day 3 (left) and the ratio of B cells expressing these IgG subclasses estimated from the flow cytometry data (right). Data are means of three biological replicates ( E ), means ± standard deviations (SDs) of two biological replicates ( A ), or three technical replicates ( C ). The data are representative of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; p values were calculated by two-way analysis of variance (ANOVA) with Tukey’s test ( A ). Figure 1—figure supplement 1—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A ) Immunoblot analysis of the indicated molecules in Igk −/− B1-8 flox/+ B cells stimulated with NP-CGG or NP-Ficoll for the indicated time periods. ( B–D ) Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd −/− Igk −/− B1-8 flox/+ B cells were stimulated with NP-Ficoll and IL-1α, IL-1β, or IFNα. The B cells were labeled with CellTrace Violet (CTV) before culture in C. ( B ) Enzyme-linked immunosorbent assay (ELISA) of IgM and IgG3 in the culture supernatants on day 5. AU, arbitrary units. ( C ) Representative flow cytometric plots of the B cells on day 3 showing percentages of IgG3 + B cells (left). The frequencies of IgG3 + cells at each cell division number (right). ( D ) qRT-PCR analysis of the circle Iγ3-Cµ and Aicda transcripts on day 2. Data are means ± standard deviations (SDs) of two ( B ) or three ( C ) biological replicates or three technical replicates ( D ). The data are representative of at least three ( A and B ) or two ( C and D ) independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated by unpaired multiple t -test ( B and C ). Figure 2—source data 1. Source data for . Figure 2—source data 2. Source data for . Figure 2—source data 3. Source data for . Figure 2—source data 4. Source data for . Figure 2—source data 5. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Western Blot, Labeling, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A and B ) Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd −/− Igk −/− B1-8 flox/+ naive B cells were cultured with medium alone (–) or NP-Ficoll. ( A ) 3 H-thymidine incorporation on day 3. ( B ) IgM concentration in the culture supernatant on day 4. ( C ) The histogram of CellTrace Violet (CTV) in cells analyzed in . ( D–G ) Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd − /− Igk −/− B1-8 flox/+ naive B cells were cultured with NP-Ficoll and LPS, R-848, or CpG ( D and E ) or LPS, R-848, or CpG alone ( F and G ). ( D and F ) IgM and IgG concentrations in the culture supernatants on day 3. ( E and G ) qRT-PCR analysis of Aicda transcripts on day 2. Data are means ± standard deviation (SD) of three ( A ) or two to three ( B ), and two ( D and F ) biological replicates, or three technical replicates ( E and G ). The data are representative of two independent experiments. *p < 0.05; ***p < 0.001; p values were calculated by unpaired multiple t -test ( A, B, and D ). Figure 2—figure supplement 1—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Cell Culture, Concentration Assay, Quantitative RT-PCR, Standard Deviation

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A ) Flow cytometric analysis of spleen cells and peritoneal cavity cells of Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd fl/fl mice. Gating strategy of follicular B cells (Fo B; CD19 + CD43 − CD21 + CD23 + ), marginal zone B cells (MZ B; CD19 + CD43 − CD21 high CD23 low ), B1a cells (CD19 + CD43 + CD5 + ), and B1b cells (CD19 + CD43 + CD5 − ) in the spleen is shown (left). The numbers of Fo B, MZ B, B1a, and B1b cells in spleen (middle) and peritoneal cavity (right) are plotted. ( B ) Enzyme-linked immunosorbent assay (ELISA) of anti-NP IgM, IgG1, and IgG3 in the sera of Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd fl/fl mice at indicated weeks after immunization with NP-CGG in alum. AU, arbitrary units. Small horizontal bars are the means of four ( A ) or six ( B ) biological replicates. Each symbol represents an individual mouse. The data are representative of three ( A ) or two ( B ) independent experiments. *p < 0.05; ****p < 0.0001; p values were calculated by unpaired multiple t- test. Figure 3—figure supplement 1—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A–C ) Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd f/f mice were immunized with NP-Ficoll. ( A ) Enzyme-linked immunosorbent assay (ELISA) of serum anti-NP IgM and IgG3 at the indicated weeks. ( B ) ELISA of serum anti-NP IgG1, IgG2b, and IgG2c at 2 weeks after immunization. ( C ) Representative flow cytometric plots of the spleen cells on day 7 after immunization. The numbers indicate the percentage of cells in each gate (left). The frequencies of IgM + , IgG2b + , and IgG3 + cells among NP-binding plasma cells (PCs; NP + CD138 + ; middle) and the numbers of such cells per 1 × 10 7 total lymphocytes (right) are plotted. ( D ) ELISA of anti-PPS3 IgM and IgG3 in the serum of Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd f/f mice at the indicated weeks after immunization with PPS3. Results are presented in AU, arbitrary units ( A, B, and D ). Small horizontal bars are the means of six to eight (A and B), five ( C ), and eight ( D ) biological replicates. Each symbol represents an individual mouse. The data are representative of three ( A and C ) or two ( B and D ) independent experiments. *p < 0.05; **p < 0.01; ****p < 0.0001; p values were calculated by unpaired multiple ( A, C, and D ) or two-tailed unpaired Welch’s t -test ( B ). Figure 3—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Clinical Proteomics, Two Tailed Test

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A and B ) B cells purified from Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi mice were labeled with CellTrace Violet (CTV) and transferred into B6 mice, which were immunized with NP-Ficoll on the next day. Spleen cells were analyzed 3 days later. ( A ) Representative flow cytometric plots of the spleen cells with the numbers indicating percentages of the cells within the neighboring gates (left). The frequency of IgG3 + cells among whole donor B cells (CD45.1 + CD19 + CD138 – ) (middle) and at each cell division number (right) are shown. ( B ) qRT-PCR analysis of the indicated transcripts in the donor B cells collected as in . ( C ) Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi B cells transduced with an empty vector (Ev) or vectors expressing AID or PKCδ were transferred into B6 mice that had been immunized with NP-Ficoll on the previous day as in . Representative flow cytometry plots of the donor cells transduced with the vectors (CD45.1 + GFP + , gated as in ) (left) and the frequency of IgG3 + cells among the CD45.1 + GFP + cells (right) on day 3 after transfer. Small horizontal bars are the means of five ( A ) or six to nine ( C ) biological replicates. Each symbol represents an individual mouse (A, middle; C ). The symbols are the means of five biological replicates (A, right). Data are means ± standard deviations (SDs) of three technical replicates pooled from five mice ( B ). The data are representative of three ( A ) or two ( B ) independent experiments or is pooled from two independent experiments ( C ). ns, not significant (p > 0.05); **p < 0.01; ****p < 0.0001; p values were calculated by unpaired multiple t -test ( A ) or one-way analysis of variance (ANOVA) with Tukey’s test ( C ). Figure 4—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Purification, Labeling, Quantitative RT-PCR, Transduction, Plasmid Preparation, Expressing, Flow Cytometry

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A ) Sorting strategy for donor B cells used in . The starting population was spleen cells of B6 mice transferred with Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi , CD45.1 + B cells and immunized with NP-Ficoll 3 days previously. CD45.1 + cells were enriched by a MACS system (left) and then donor B cells (CD45.1 + CD19 + CD138 − ) were further sorted by flow cytometry (right). ( B and C ) Strategy for retrovirus (Rv) transduction and the analysis of the TI-2 response (B, left). B cells collected from Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi mice immunized with NP-Ficoll 1 day previously were infected with Rv and cultured for 1 day. Some Rv-transduced cells were further cultured in medium for 6 hr and analyzed by flow cytometry. Representative data of the cells transduced with pMXs-IRES-GFP empty vector (Ev) is shown (B, right). Other Rv-transduced cells were transferred into B6 mice that had been immunized with NP-Ficoll on the previous day. Spleen cells of recipient mice were analyzed or used for cell sorting 3 or 4 days later. Gating strategy of donor cells transduced with vectors (CD45.1 + GFP + ) and the expression profile of IgG3 are shown (C, left). After the enrichment of CD45.1 + cells by a MACS system, the vector-transduced donor B cells (CD45.1 + GFP + CD19 + CD138 – ) were sorted as shown (C, right). The data are representative of at least three independent experiments.

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Flow Cytometry, Transduction, Infection, Cell Culture, Plasmid Preparation, FACS, Expressing

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: ( A ) qRT-PCR analysis of the transcripts of the indicated genes in Prkcd +/+ Cd19 cre/+ B1-8 hi or Prkcd f/f Cd19 cre/+ B1-8 hi donor B cells (CD45.1 + CD19 + CD138 − ) purified as in from the recipient mice immunized with NP-Ficoll 3 days previously. Shown is the relative expression of each gene in Prkcd f/f Cd19 cre/+ cells to that in Prkcd +/+ Cd19 cre/+ cells. ( B ) qRT-PCR analysis of Batf transcripts in Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd −/− Igk −/− B1-8 flox/+ B cells cultured with medium alone (–) or with the indicated stimuli for 2 days. ( C ) Immunoblot analysis in Prkcd + /+ Igk −/− B1-8 flox/+ or Prkcd −/− Igk −/− B1-8 flox/+ B cells stimulated with NP-Ficoll for the indicated times. ( D and E ) B1-8 hi B cells transduced with knockdown vectors for luciferase (shControl) or BATF (shBATF) were transferred into B6 mice that had been immunized with NP-Ficoll on the previous day as in , and their spleen cells were analyzed on day 4 after transfer. ( D ) qRT-PCR analysis of Aicda and Batf transcripts in the vector-transduced donor B cells (CD45.1 + GFP + CD19 + CD138 − ) collected as in . ( E ) Representative flow cytometric plots of the transduced donor cells with the numbers indicating the percentage of IgG3 + cells (left) and the frequency of the IgG3 + cells among such cells (right) gated as in . ( F and G ) Prkcd f/f Cd19 cre/+ B1-8 hi B cells transduced with Ev or vectors expressing BATF or PKCδ were transferred into B6 mice that had been immunized with NP-Ficoll on the previous day, and their spleen cells were analyzed 3 days after transfer. ( F ) qRT-PCR analysis of Aicda transcripts in the vector-transduced donor B cells. ( G ) Representative flow cytometric plots of the transduced donor cells with the numbers indicating the percentage of IgG3 + cells (left) and the frequency of the IgG3 + cells among such cells (right). Data are means ± standard deviations (SDs) of three technical replicates ( A, B, D, and F ). Samples were pooled from five to eight mice ( D and F ). Small horizontal bars are the means of four ( E ) or eight to nine ( G ) biological replicates. Each symbol represents an individual mouse ( E and G ). The data are representative of two independent experiments ( A–F ) or are pooled from two independent experiments ( G ). ***p < 0.001; ****p < 0.0001; p values were calculated by two-tailed unpaired Student’s t -test ( E ) or one-way analysis of variance (ANOVA) with Tukey’s test ( G ). Figure 5—source data 1. Source data for . Figure 5—source data 2. Source data for . Figure 5—source data 3. Source data for . Figure 5—source data 4. Source data for . Figure 5—source data 5. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Quantitative RT-PCR, Purification, Expressing, Cell Culture, Western Blot, Transduction, Knockdown, Luciferase, Plasmid Preparation, Two Tailed Test

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: Cd19 cre/+ Prkcd +/+ and Cd19 cre/+ Prkcd f/f mice were cohoused for at least 4 weeks and treated with 3 % dextran sodium sulfate (DSS) for 7 days. ( A ) Serum IgM, IgG1, IgG2b, and IgG3 against fecal bacteria after cohousing were quantified by enzyme-linked immunosorbent assay (ELISA). AU, arbitrary units. ( B ) Colony-forming unit (CFU) of aerobes and anaerobes in the blood 7 days after DSS treatment. ( C ) Kaplan–Meier survival plot of 16 ( Cd19 cre/+ Prkcd +/+ ) and 12 ( Cd19 cre/+ Prkcdf f/f ) mice at indicated days. Small horizontal bars are the means of 7 ( A ), or 15 ( Cd19 cre/+ Prkcd +/+ ) or 11 ( Cd19 cre/+ Prkcdf f/f ) in ( B ), biological replicates. Data were obtained from 16 ( Cd19 cre/+ Prkcd +/+ ) or 12 ( Cd19 cre/+ Prkcdf f/f ) mice in ( C ). Each symbol represents an individual mouse ( A and B ). The data are representative of two independent experiments ( A ) or pooled from two independent experiments ( B and C ). **p < 0.01; ***p < 0.001; p values were calculated by two-tailed unpaired Welch’s t -test ( A ), unpaired multiple t -test ( B ), or log-rank test ( C ). Figure 6—source data 1. Source data for .

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Bacteria, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: eLife

Article Title: Protein kinase Cδ is essential for the IgG response against T-cell-independent type 2 antigens and commensal bacteria

doi: 10.7554/eLife.72116

Figure Lengend Snippet: B-cell receptor (BCR) stimulation with a TI-2 antigen (Ag) activates PKCδ signaling and induces the expression of BATF, which works cooperatively with secondary signals induced by IL-1α/β, IFNα, or TLR ligands and drives the transcription of activation-induced cytidine deaminase (AID) to induce IgG CSR.

Article Snippet: Antibody , Goat F(ab')2 Anti-Mouse IgG2b, Human ads-FITC (Goat Polyclonal) , Southern Biotech , Cat# 1092-02 , Flow cytometry (1/300).

Techniques: Expressing, Activation Assay

Journal:

Article Title: Mycobacterium bovis BCG-Infected Mice Are More Susceptible to Staphylococcal Enterotoxin B-Mediated Toxic Shock than Uninfected Mice despite Reduced In Vitro Splenocyte Responses to Superantigens †

doi: 10.1128/IAI.70.8.4148-4157.2002

Figure Lengend Snippet: Infection with BCG results in accumulation and not deletion of superantigen-responsive CD4+ or CD8+ T cells. Unfractionated splenocytes (5 × 105 cells/tube) from SEB-exposed mice (7 days postinjection) (A) and PBS-injected controls or day-16-infected mice and PBS-injected controls (B) were stained with FITC-labeled anti-mouse Vβ8 TCR (A and B) and either PE-labeled anti-mouse CD4 or anti-mouse CD8. The percentages of Vβ8+ CD4+ T cells (white bars) or Vβ8+ CD8+ T cells (black bars) in the spleen were determined by flow cytometry. Means ± standard errors of the means of three experiments are shown. ∗, P < 0.05.

Article Snippet: In some experiments, splenocytes that were cultured for 24 or 72 h with SEB or BCG antigen were stained with FITC-conjugated rat anti-mouse CD4 (YTS 191.1) and rat anti-mouse CD8α (IM7.8.1; both from Cedarlane) and counterstained with propidium iodide (PI) (10 μg/ml; Sigma) to enumerate viable cells.

Techniques: Infection, Injection, Staining, Labeling, Flow Cytometry

Journal:

Article Title: Mycobacterium bovis BCG-Infected Mice Are More Susceptible to Staphylococcal Enterotoxin B-Mediated Toxic Shock than Uninfected Mice despite Reduced In Vitro Splenocyte Responses to Superantigens †

doi: 10.1128/IAI.70.8.4148-4157.2002

Figure Lengend Snippet: BCG infectious dose correlates with increased expression of the T-cell activation markers CD44, IL-2Rα, and IL-2Rβ. Unfractionated splenocytes (5 × 105 cells/tube) from each type of spleen were stained with FITC-labeled anti-mouse CD44, anti-mouse CD25/IL-2Rα, or anti-mouse CD122/IL-2Rβ and PE-labeled anti-mouse CD4 or anti-mouse CD8α. The percentages of CD4+ CD44hi, IL-2Rαhi, or IL-2Rβhi CD4+ (white bars) or CD8+ cells (black bars) in the spleen were determined by flow cytometry. Means ± standard errors of the means of three experiments are shown. ∗, P < 0.05; ∗∗, P < 0.01. Purified CD4+ T cells from the spleens of BCG HD-infected mice (day 16) were stained with FITC anti-mouse CD44 and were sorted in an EPICS Elite ESP (Beckman Coulter) into CD44low (naïve) and CD44hi (memory/effector) populations. Sorted cells were cocultured at 105 cells/well either with bone marrow-derived dendritic cells (5 × 104 cells/well) in the presence or absence of SEB (10 μg/ml) or with M12 B-lymphoma cells (5 × 104 cells/well) in the presence or absence of BCG-Ag (10 μg/ml). IFN-γ levels were determined as described in Materials and Methods. Mean ± standard deviation of triplicate wells is shown.

Article Snippet: In some experiments, splenocytes that were cultured for 24 or 72 h with SEB or BCG antigen were stained with FITC-conjugated rat anti-mouse CD4 (YTS 191.1) and rat anti-mouse CD8α (IM7.8.1; both from Cedarlane) and counterstained with propidium iodide (PI) (10 μg/ml; Sigma) to enumerate viable cells.

Techniques: Expressing, Activation Assay, Staining, Labeling, Flow Cytometry, Purification, Infection, Derivative Assay, Standard Deviation

Journal:

Article Title: Mycobacterium bovis BCG-Infected Mice Are More Susceptible to Staphylococcal Enterotoxin B-Mediated Toxic Shock than Uninfected Mice despite Reduced In Vitro Splenocyte Responses to Superantigens †

doi: 10.1128/IAI.70.8.4148-4157.2002

Figure Lengend Snippet: BCG infection induces potent APCs that stimulate rather than inhibit IFN-γ production to SEB and BCG-Ag by purified CD4+ T cells. Sixteen days after injection of mice with PBS or BCG LD or BCG HD, adherent cells were obtained from bulk splenocyte suspensions by panning as described in Materials and Methods. Adherent cell populations (5 × 105 cells/well) were cocultured with purified CD4+ T cells (105 cells/well) from the same mice in the presence of either SEB (10 μg/ml) (A) or BCG-Ag (10 μg/ml) (B). (C) KLH-specific HDK1 type 1 clone was cocultured at 105 cells/well with different numbers of unfractionated splenocytes from PBS control mice or BCG LD- or HD-infected mice in the presence of KLH (10 μg/ml). IFN-γ levels were determined as described in the Fig. ​Fig.11 legend. Mean ± standard deviation of triplicate wells is shown. (D) BCG-infected spleens have more MHC class II+ and Mac1+ cells. Unfractionated splenocytes (5 × 105 cells/tube) from mice that were infected for 2 weeks with BCG LD- or BCG HD-infected mice or PBS-injected controls were stained with FITC-labeled anti-mouse I-Ad/I-Ed/MHC class II and PE-labeled anti-mouse Mac1/CD11b. The percentages of MHC class II+ (white bars) and Mac1+ (black bars) APCs in the spleen were determined by flow cytometry. Means ± standard errors of the means of three experiments are shown.

Article Snippet: In some experiments, splenocytes that were cultured for 24 or 72 h with SEB or BCG antigen were stained with FITC-conjugated rat anti-mouse CD4 (YTS 191.1) and rat anti-mouse CD8α (IM7.8.1; both from Cedarlane) and counterstained with propidium iodide (PI) (10 μg/ml; Sigma) to enumerate viable cells.

Techniques: Infection, Purification, Injection, Standard Deviation, Staining, Labeling, Flow Cytometry

Journal:

Article Title: Mycobacterium bovis BCG-Infected Mice Are More Susceptible to Staphylococcal Enterotoxin B-Mediated Toxic Shock than Uninfected Mice despite Reduced In Vitro Splenocyte Responses to Superantigens †

doi: 10.1128/IAI.70.8.4148-4157.2002

Figure Lengend Snippet: HD splenocytes have a decreased percentage of T cells and show reduced viability early after culture with BCG-Ag and SEB. (A) Unfractionated splenocytes (5 × 105 cells/tube) from mice that were infected for 2 or 3 weeks with BCG LD or BCG HD or from PBS-injected controls were stained with FITC-labeled anti-mouse CD4 and PE-labeled anti-mouse CD8α. The percentages of CD4+ (white bars) or CD8+ cells (black bars) in the spleen were determined by flow cytometry. Means ± standard errors of the means of three experiments are shown. ∗, P < 0.05; ∗∗, P < 0.01. (B) Unfractionated splenocytes were subsequently stimulated with BCG-Ag (10 μg/ml) or SEB (10 μg/ml) at 5 × 105 cells/well in parallel cultures. At 24 h, cells from triplicate wells were pooled and stained with both FITC-labeled anti-mouse CD4 and anti-mouse CD8α and were counterstained with PI (10 μg/ml). The total percentage of nonviable (PI+) T cells was determined by flow cytometry.

Article Snippet: In some experiments, splenocytes that were cultured for 24 or 72 h with SEB or BCG antigen were stained with FITC-conjugated rat anti-mouse CD4 (YTS 191.1) and rat anti-mouse CD8α (IM7.8.1; both from Cedarlane) and counterstained with propidium iodide (PI) (10 μg/ml; Sigma) to enumerate viable cells.

Techniques: Infection, Injection, Staining, Labeling, Flow Cytometry

Journal: Oncotarget

Article Title: Oncogenic KIT-induced aggressive systemic mastocytosis requires SHP2/PTPN11 phosphatase for disease progression in mice

doi:

Figure Lengend Snippet: A. Representative images of spleens isolated from DBA/2 mice at day 13 following retro-orbital injection of saline (sham), or P815-NT cells, or P815-KD cells (5 × 10 3 in 100 μl; 3-4 mice/group; 3 experiments). B. Graph depicts spleen weights from saline-injected sham mice (n=3), or mice injected with P815-NT or P815–KD cells (n=11) at day 13 (mean ± SD; * indicates significant difference between groups, p <0.05). C. Splenocytes isolated from mice described above were analyzed by flow cytometry. Representative histograms are shown for splenocytes stained with FITC-Gr1/PE-CD11b/PEcy5-B220, with mean values shown for single and double positive populations (n=3; results are representative of 3 independent experiments). D. Graph depicts absolute cell numbers (x10 6 ) for Gr1 + , CD11b + , and B220 + cells in spleen from mice described above (* indicates significant difference between NT and KD, p <0.05). E. Representative histograms are shown for splenocytes stained with PE-KIT/PEcy5-CD45, with mastocytoma cells identified as double positives, from mice described above. F. Graph depicts the absolute mastocytoma cell numbers in spleen (mean ± SD; n=10; * indicates significant difference between NT and KD, p <0.05).

Article Snippet: [ ] Antibodies for flow cytometry included: PE-conjugated anti-mouse CD117 (2B8; BD Bioscience), PE/cy5-conjugated anti-mouse CD45 (eBioscience), PE conjugated anti-mouse CD11b (M1/70; Biolegend), PE/cy5-conjugated anti-mouse CD45R (B220) (RA3-6B2; Biolegend), FITC anti-mouse Gr1 (RB6-8C5; Cedarlane).

Techniques: Isolation, Injection, Saline, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: Oncogenic KIT-induced aggressive systemic mastocytosis requires SHP2/PTPN11 phosphatase for disease progression in mice

doi:

Figure Lengend Snippet: A. BM cells were isolated from DBA/2 mice at day 13 following retro-orbital injection of saline (sham), or P815-NT cells, or P815-KD cells. BM cells were stained with FITC-Gr1/PE-CD11b/PEcy5-B220 and analyzed by flow cytometry. Graph depicts absolute cell numbers (x10 6 ) for Gr1 + , CD11b + , and B220 + cells in BM from sham (n=3), or mice injected with P815-NT or P815-KD cells (mean ± SD, n=11; * indicates significant difference between NT and KD, p <0.05). B. Representative histograms are shown for BM cells stained with PE-KIT/PEcy5-CD45, with mastocytoma cells identified as double positives, from mice described above. C. Graph depicts the absolute mastocytoma cell numbers in BM (mean ± SD; n=11; * indicates significant difference between NT and KD, p <0.05).

Article Snippet: [ ] Antibodies for flow cytometry included: PE-conjugated anti-mouse CD117 (2B8; BD Bioscience), PE/cy5-conjugated anti-mouse CD45 (eBioscience), PE conjugated anti-mouse CD11b (M1/70; Biolegend), PE/cy5-conjugated anti-mouse CD45R (B220) (RA3-6B2; Biolegend), FITC anti-mouse Gr1 (RB6-8C5; Cedarlane).

Techniques: Isolation, Injection, Saline, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: Oncogenic KIT-induced aggressive systemic mastocytosis requires SHP2/PTPN11 phosphatase for disease progression in mice

doi:

Figure Lengend Snippet: A. Single cell suspensions from peripheral blood were isolated from DBA/2 mice at day 13 following retro-orbital injection of saline (sham), or P815-NT cells, or P815-KD cells. Following lysis of erythrocytes, cells were stained with FITC-Gr1/PE-CD11b/PEcy5-B220, and analyzed by flow cytometry. Graph depicts absolute cell numbers (x10 6 ) for Gr1 + , CD11b + , and B220 + cells in peripheral blood from sham (n=3), or mice injected with P815-NT or P815-KD cells (mean ± SD, n=9 from 3 separate experiments). B. Representative histograms are shown for peripheral blood cells stained with PE-KIT/PEcy5-CD45, with mastocytoma cells identified as double positives, from mice analyzed in 3 independent experiments as described above. C. Graph depicts the absolute mastocytoma cell numbers in peripheral blood (mean ± SD; n=9 from 3 separate experiments; * indicates significant difference between NT and KD, p <0.05).

Article Snippet: [ ] Antibodies for flow cytometry included: PE-conjugated anti-mouse CD117 (2B8; BD Bioscience), PE/cy5-conjugated anti-mouse CD45 (eBioscience), PE conjugated anti-mouse CD11b (M1/70; Biolegend), PE/cy5-conjugated anti-mouse CD45R (B220) (RA3-6B2; Biolegend), FITC anti-mouse Gr1 (RB6-8C5; Cedarlane).

Techniques: Isolation, Injection, Saline, Lysis, Staining, Flow Cytometry

Journal:

Article Title: Mycobacterium bovis BCG-Infected Mice Are More Susceptible to Staphylococcal Enterotoxin B-Mediated Toxic Shock than Uninfected Mice despite Reduced In Vitro Splenocyte Responses to Superantigens †

doi: 10.1128/IAI.70.8.4148-4157.2002

Figure Lengend Snippet: BCG infectious dose correlates with increased expression of the T-cell activation markers CD44, IL-2Rα, and IL-2Rβ. Unfractionated splenocytes (5 × 105 cells/tube) from each type of spleen were stained with FITC-labeled anti-mouse CD44, anti-mouse CD25/IL-2Rα, or anti-mouse CD122/IL-2Rβ and PE-labeled anti-mouse CD4 or anti-mouse CD8α. The percentages of CD4+ CD44hi, IL-2Rαhi, or IL-2Rβhi CD4+ (white bars) or CD8+ cells (black bars) in the spleen were determined by flow cytometry. Means ± standard errors of the means of three experiments are shown. ∗, P < 0.05; ∗∗, P < 0.01. Purified CD4+ T cells from the spleens of BCG HD-infected mice (day 16) were stained with FITC anti-mouse CD44 and were sorted in an EPICS Elite ESP (Beckman Coulter) into CD44low (naïve) and CD44hi (memory/effector) populations. Sorted cells were cocultured at 105 cells/well either with bone marrow-derived dendritic cells (5 × 104 cells/well) in the presence or absence of SEB (10 μg/ml) or with M12 B-lymphoma cells (5 × 104 cells/well) in the presence or absence of BCG-Ag (10 μg/ml). IFN-γ levels were determined as described in Materials and Methods. Mean ± standard deviation of triplicate wells is shown.

Article Snippet: For flow cytometry analysis, splenocytes (5 × 10 5 ) were stained with all or some of the following MAbs (all at 1:100 dilutions in R10A): fluorescein isothiocyanate (FITC)-conjugated mouse anti-mouse Vβ8 TCR (F23.1), FITC-conjugated rat anti-mouse CD25 (IL-2 Rα; 7D4), FITC-conjugated rat anti-mouse CD122 (IL-2 Rβ; TM-β1), FITC-conjugated rat anti-mouse I-A d /I-E d (2G9) (all from PharMingen), or FITC-conjugated rat anti-mouse CD44 (IM7.8.1; Cedarlane Laboratories, Hornby, Canada).

Techniques: Expressing, Activation Assay, Staining, Labeling, Flow Cytometry, Purification, Infection, Derivative Assay, Standard Deviation