Expression of IL-2 by stimulated CD3+ and CD3-human PBMC. Human PBMC were stimulated for 6 h with PMA (Sigma, Cat. #P-8139) and calcium ionophore A23187 (Sigma, Cat. #C-9275), in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were stained with FITC Mouse Anti-Human CD3 (Cat. No. 555332), fixed, permeabilized, and then stained with 0.25 µg of APC Rat Anti-Human IL-2 (Cat. No. 554567/561054; left panel). To demonstrate specificity of staining, the binding of APC-MQ1-17H12 was blocked by the preincubation of the conjugated antibody with excess recombinant human IL-2 (1.0 µg, Cat. No. 554603; middle panel), and by preincubation of the fixed/permeabilized cells with an excess of Purified Rat Anti-Human IL-2 (10 µg, Cat. No. 554563; right panel) prior to staining with the APC-MQ1-17H12 antibody. 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 specificity controls (right panel). This APC-conjugated reagent can be used in any flow cytometer equipped with a dye, HeNE or red diode laser. These include the dual laser FACStarPLUS™, FACS Vantage™ or FACSCalibur™.