Expression of IL-6 by stimulated CD14+ human monocytes. Human PBMC were stimulated for 6 hours with LPS (100 ng/ml final concentration) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The PBMC were harvested, stained with PE-mouse anti-human CD14 monoclonal antibody (PE-M5E2, Cat. No. 555398), fixed, permeabilized, and subsequently stained with either 0.25 µg of FITC-Rat IgG1 isotype control immunoglobulin (FITC-R3-34; Cat. No. 554684; middle panel) or with 0.25 µg of FITC-rat anti-human IL-6 antibody (FITC-MQ2-13A5; Cat. No. 554544; right panel), following Pharmingen's staining protocol. Left panel portrays autofluorescence detected by the FL1 PMT versus CD14-PE staining. The data reflect gating on monocytes, based on forward and side scattered light signals. To demonstrate specificity of staining, the binding by FITC-MQ2-13A5 antibody was blocked by each of the following: 1) preincubation of the fluorochrome-conjugated antibody with recombinant human IL-6 (Cat. No. 550071; data not shown) and by 2) preincubation of the fixed/permeabilized cells with unlabeled MQ2-13A5 antibody (Cat. No. 554542; data not shown) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the ligand blocking and unlabeled antibody blocking specificity controls.