Expression of IL-2 by stimulated CD4+ and CD4- BALB/c spleen cells. Splenocytes from 6 month-old BALB/C mice were stimulated for 5 hours with hamster anti-mouse CD3 (2 mg/ml final concentration; Cat. No.553057, clone 145-2C11) and hamster anti-mouse CD28 (2 mg/ml final concentration; Cat. No. 553294, clone 37.51) antibodies in the presence of BD GolgiStop™ (3 µM final concentration; Cat. No. 554724). The cells were harvested, stained with 0.06 µg of PE-conjugated rat anti-mouse CD4 (PE-RM4-5, Cat. No. 553049), fixed, permeabilized, and subsequently stained with 0.06 µg of FITC-conjugated rat anti-mouse IL-2 antibody (FITC-JES6-5H4, Cat. No. 554427) using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding by FITC-JES6-5H4 was blocked by each of the following: 1) preincubation of the conjugated antibody with recombinant mouse IL-2 (0.12 mg, Cat. No. 550069; middle panel), and by 2) preincubation of the fixed/permeabilized cells with excess unlabelled JES6-5H4 antibody 2.5 mg; Cat. No. 554425; right panel) prior to staining with the FITC-JES6-5H4 antibody. 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 controls. A suitable rat IgG2b isotype control for assessing the level of background staining on fixed/permeabilized mouse cells is Cat. No. 556923; use at comparable concentrations to antibody of interest.