Expression of IL-2 by stimulated CD3+ human PBMC. Human PBMC were stimulated for 6 hours with PMA (Sigma) and calcium ionophore A23187 (Sigma) in the presence of GolgiStop™ (2 mM final concentration; Cat. No. 554714). The PBMC were stained with PE-Cy5-anti-CD3 (PE-CY5 UCHT1, Cat. No 555334), fixed, permeabilized, and subsequently stained with 0.25 mg of FITC-Rat IgG2a, κ anti-human IL-2 antibody (FITC-MQ1-17H12, Cat. No 554565; left panel) or 0.25 mg FITC-R35-95 isotype control immunoglobulin (FITC-R35-95, Cat. No. 554688; right panel) using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of FITC-MQ1-17H12 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant human IL-2 (Cat. No.554603; data not shown), and by preincubation of the fixed/permeabilized cells with an excess of the unlabelled MQ1-17H12 antibody (Cat. No. 554563; data not shown) prior to staining with the FITC-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 and unlabelled antibody blocking specificity controls.