Expression of IL-10 by stimulated CD4+ Balb/c spleen cells. Purified splenic CD4+ cells from 6 month old BALB/c mice were stimulated with plate-bound anti-CD3 (clone 145-2C11, Cat. No. 553058 at 25 µg/ml) and soluble anti-mouse CD28 (clone 37.51, Cat. No. 553294 at 2 µg/ml) for 2 days in culture together with recombinant mouse IL-2 (10 ng/ml, Cat. No. 550069) and recombinant mouse IL-4 (1 ng/ml, Cat. No. 550067), followed by a 3 day incubation with only IL-2 and IL-4. This was followed by a 5 hour stimulation with plate-bound anti-CD3 (25 µg/ml) and anti-mouse CD28 (2 µg/ml) in the presence of GolgiStop™ (Cat. No. 554724). The cells were then stained with 0.06 µg of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553046) and 0.12 µg of PE-conjugated rat anti-mouse IL-10 antibody (PE-JES5-16E3, Cat. No. 554467) by using BD Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of PE-JES5-16E3 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-10 (0.12 µg, Cat. No.550070; middle panel), and by preincubation of the fixed and permeabilized cells with an excess of the unlabelled JES5-16E3 mAb (3.6 µg, Cat. No. 554464; right panel). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (middle panel) and unlabelled antibody blocking (right panel) specificity controls. A suitable rat IgG2b isotype control for assessing levels of background staining on fixed/permeabilized mouse cells is PE-R35-38 (Cat. No. 556925); use at comparable concentrations to antibody of interest.