Expression of GM-CSF by stimulated human peripheral blood mononuclear cells (PBMC). Ficoll™-separated human PBMC were stimulated with Purified NA/LE Mouse Anti-Human CD3 (1 µg/ml final concentration; Cat. No. 555329), 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. Finally, the cells were harvested and stimulated for 6 hours with PMA (50 ng/ml final concentration;  Sigma, Cat. #P-8139) and calcium ionophore A23187 (250 ng/ml final concentration; Sigma, Cat. #C-9275) in the presence of  BD GolgiStop™ (2 µM final concentration; Cat. No. 554724). The cells were harvested, stained with PE-Cy™7 Mouse Anti-Human CD4 (Cat. No. 557852), fixed, permeabilized, and subsequently stained with 0.25 µg of PE Rat anti-Human GM-CSF (Cat. No. 554507) antibody by using the intracellular staining protocol (see Left panel). To demonstrate specificity of staining, the binding of PE Rat Anti-Human GM-CSF was blocked by preincubation of the antibody conjugate with recombinant human GM-CSF (0.1 µg; Cat. No. 550068; see Center panel) and by preincubation of the fixed/permeabilized cells with Purified Rat Anti-Human GM-CSF (5 µg; Cat. No. 554503; see Right panel) prior to staining. The quadrant markers for the bivariate dot plot were set based on the autofluorescence control and verified using the ligand-blocking and unlabeled antibody blocking controls.