Expression of IFN-γ by stimulated CD8+ and CD8- BALB/c spleen cells. Splenocytes from 6 month old BALB/c mice were cultured for 3 days in the presence of SEB (2 µg/ml; Sigma, Cat. No. S-4881), then restimulated for 5 hour with hamster anti-mouse CD3 (2 µg/ml, 145-2C11, Cat. No. 553057) and hamster anti-mouse CD28 (2 µg/ml, 37.51, Cat. No, 553294) antibodies in the presence of 2 µM GolgiStop™ (aka, monensin; Cat. No. 554724). The splenocytes were harvested, stained with 0.06 µg of FITC rat anti-mouse CD8 (FITC 53-6.7, Cat. No. 553030), fixed, permeabilized, and subsequently stained with 0.06 µg of PE rat anti-mouse IFN-γ (PE-XMG1.2, Cat. No. 554412, left panel) by using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding by the PE-XMG1.2 antibody was blocked by preincubation of the fixed/permeabilized cells with unlabeled XMG1.2 antibody (5.0 µg; see right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.