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Expression of TNF by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated for 6 hours 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. 554715). The PBMC were stained with PE-Cy™5 Mouse Anti-Human CD3 (Cat. No. 555334), fixed, permeabilized, and subsequently stained with 0.25 µg of FITC Mouse Anti-Human TNF (Cat. No. 554512/562082/552889, left panel). To demonstrate specificity of staining, the binding of FITC Mouse Anti-Human TNF was blocked by the preincubation of the fluorochrome-conjugated antibody with recombinant human TNF (5 µg, Cat. No. 554618; middle panel), and by preincubation of the fixed/permeabilized cells with Purified Mouse Anti-Human TNF (5 µg, Cat. No. 554510; right panel) prior to staining with the FITC Mouse Anti-Human TNF. 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.