Expression of IL-6 by stimulated BALB/c cells. BALB/c bone marrow cells were cultured for 2 weeks in the presence of recombinant mouse GM-CSF (20 ng/ml final concentration; Cat. No. 554586). The cells were washed and stimulated with recombinant mouse IFN-γ (10 ng/ml final concentration; Cat. No. 554587) for 4 hours followed by an overnight stimulation with LPS (1 µg/ml final concentration; Sigma, Cat. #L-8272) and GolgiPlug™ (1 µg/ml final concentration; Cat. No. 555029; aka Brefeldin A). Adherent cells were incubated with 1x trypsin EDTA at 37°C for 10 minutes and gently dislodged by pipetting for cell harvest. Nonspecific binding of cell surface antigens was blocked by incubation of the cells with Fc Block™ (10 µg/ml final concentration; Cat. No. 553142). The cells were fixed , permeabilized then subsequently stained with 0.06 µg of PE-rat anti-mouse IL-6 antibody (PE-MP5-20F3, Cat. No. 554401) by using Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding by the PE-MP5-20F3 antibody was blocked by preincubation of the PE-MP5-20F3 antibody with recombinant mouse IL-6 (0.12 µg; Cat. No. 554582; right panel) and by preincubation of the fixed/permeabilized cells with unlabeled MP5-20F3 antibody (5 µg; Cat. No. 554400; right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.