Expression of IL-2 by stimulated CD4+ and CD4- BALB/c spleen cells. Splenocytes from 6 month old BALB/C mice were stimulated for 15 hr with hamster anti-mouse CD3 (25 µg/ml, clone 145-2C11, Cat. No. 553057) and hamster anti-mouse CD28 (2 µg/ml, clone 37.51, Cat. No. 553294) antibodies in the presence of BD GolgiPlug™ (Cat. No. 555029). The splenocytes were harvested, stained with 0.06 µg of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047), fixed, permeabilized, and subsequently stained with 0.25 µg of APC-conjugated rat anti-mouse IL-2 antibody (APC-JES6-5H4, Cat. No. 554429) by using the BD Pharmingen staining protocol (left panel). To demonstrate specificity of staining, the staining by APC-JES6-5H4 was blocked by each of the following:  1) preincubation of the conjugated antibody with recombinant mouse IL-2 (0.25 µg, Cat. No. 550069; middle panel) and by 2) preincubation of the fixed/permeabilized cells with unlabelled JES6-5H4 mouse antibody (5.0 µg; Cat. No. 554425) prior to staining with the APC-JES6-5H4 (right panel). 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. This APC-conjugated reagent can be used in any flow cytometer equipped with a a dye, HeNE or red diode laser. These include the dual laser FACStarPLUS™, FACS Vantage™ or FACSCalibur™.