Detection of IL-5 expression by stimulated human CD4+ T cells. Isolated human CD4+ cells were stimulated with immobilized anti-human CD3 (1 µg/ml final concentration; UCHT1, Cat. No. 555329), soluble anti-human CD28.2 antibody (20 ng/ml final concentration; Cat. No. 555725), recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and recombinant human IL-4 (10 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and recombinant human IL-4 for 3 days. The cells were then harvested and restimulated for 6 hours with PMA (Sigma, Cat. No. P-8139) and calcium ionophore A23187 (Sigma, Cat. No. C-9275) in the presence of BD GolgiStop™ (2 µM final concentration; Cat. No. 554724). Finally, the cells were harvested, stained with 0.25 µg of PE-Cy5 anti-CD4 (PE-CY5-RPA-T4, Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.12 µg of PE-conjugated rat anti-human IL-5 (PE-JES1-39D10, Cat. No. 554489) by using BD Biosciences Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding of PE-JES1-39D10 antibody was blocked by the preincubation of the conjugated antibody with recombinant human IL-5 (0.1 µg; Cat. No. 554606; middle panel), and by preincubation of the fixed/permeabilized cells with unlabeled JES1-39D10 antibody (5 µg, Cat. No. 554487; right panel) prior to staining with the PE-JES1-39D10 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control and verified with the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.