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 (145-2C11, Cat. No. 553057 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 recombinant 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 2 µM monensin (BD GolgiStop™ Cat. No. 554724). The cells were harvested, stained with PE Rat anti-Mouse CD4 (RM4-5, Cat. No. 553049), fixed, permeabilized, and subsequently stained with FITC Rat anti-Mouse IL-10 (JES5-16E3, Cat. No. 562037) by using the BD Pharmingen staining protocol. To demonstrate specificity of staining, the binding of FITC-JES5-16E3 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-10 (Cat. No. 550070; middle panel), and by preincubation of the fixed/permeabilized cells with an excess of the unlabelled JES5-16E3 antibody (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 background staining on fixed/permeabilized mouse cells is FITC-A95-1 (Cat. No. 556923); use at comparable concentrations to antibody of interest.