参考图片
Expression of IFN-γ by stimulated CD8+ and CD8- BALB/c spleen cells. BALB/c spleen cells were cultured for 72h in medium containing Staphylococcus aureus entertoxin B (SEB, 2 µg/ml; Sigma Cat. No. S-4881), recombinant mouse IL-2 (10 U/ml, BD Cat. No. 550069) and recombinant mouse IL-4 (2 ng/ml, BD Cat. No. 550067). The cells were harvested and restimulated for 5 h with immobilized anti-CD3 (145-2C11, BD Cat. No. 553057 at 10 µg/ml) and anti-CD28 (clone 37.51, BD Cat. No. 553294 at 2 µg/ml) antibodies in the presence of BD GolgiStop™ (2 µM final concentration, Cat. No. 554724). The splenocytes were stained with 0.25 µg of PE rat anti-mouse CD8 (PE-53-6.7, Cat. No. 553033), fixed permeabilized, and subseqently stained with 0.1 µg of FITC rat anti-mouse IFN-γ (FITC-XMG1.2, left panel). To demonstrate specificity of staining, the binding by FITC-XMG1.2 was blocked by preincubation of the fixed/permeabilized cells with excess unlabeled XMG1.2 mAb (5 µg; Cat. No. 554409, right panel) prior to staining with the FITC-XMG1.2. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabelled antibody blocking specificity control.
Expression of IFN-γ by stimulated CD8+ and CD8- BALB/c spleen cells. BALB/c spleen cells were cultured for 72h in medium containing Staphylococcus aureus entertoxin B (SEB, 2 µg/ml; Sigma Cat. No. S-4881), recombinant mouse IL-2 (10 U/ml, BD Cat. No. 550069) and recombinant mouse IL-4 (2 ng/ml, BD Cat. No. 550067). The cells were harvested and restimulated for 5 h with immobilized anti-CD3 (145-2C11, BD Cat. No. 553057 at 10 µg/ml) and anti-CD28 (clone 37.51, BD Cat. No. 553294 at 2 µg/ml) antibodies in the presence of BD GolgiStop™ (2 µM final concentration, Cat. No. 554724). The splenocytes were stained with 0.25 µg of PE rat anti-mouse CD8 (PE-53-6.7, Cat. No. 553033), fixed permeabilized, and subseqently stained with 0.1 µg of FITC rat anti-mouse IFN-γ (FITC-XMG1.2, left panel). To demonstrate specificity of staining, the binding by FITC-XMG1.2 was blocked by preincubation of the fixed/permeabilized cells with excess unlabeled XMG1.2 mAb (5 µg; Cat. No. 554409, right panel) prior to staining with the FITC-XMG1.2. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabelled antibody blocking specificity control.
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