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