Expression of IL-12 by mouse bone marrow-derived macrophages. Bone marrow cells from 6 month old BALB/c mice were cultured for 10 days in mouse GM-CSF (40 ng/ml final concentration; Cat. No. 554586). Adherent cells were washed and treated for ~14 hours with mouse IFN-γ (10 ng/ml final concentration; Cat. No. 554587); subsequently LPS (1 µg/ml final concentration; Sigma) and GolgiStop™ (2 µM final concentration Cat. No. 554724) were added to cultures for an additional 5 hours. Adherent cells were washed and then incubated with 1x trypsin EDTA at 37°C. for 15 minutes and gently dislodged by pipetting. Nonspecific surface binding was blocked by incubation of cells with purified polyclonal normal mouse immunoglobulin. Cells were then fixed, permeabilized, and non-specific binding to intracellular antigens was blocked using PBS/2%BSA/0.1% saponin. Cells were then stained with 0.06 µg of PE-conjugated rat anti-mouse IL-12 (p40/p70) antibody (PE-C15.6, Cat. No. 554479; see left panel) using the BD Pharmingen™ staining protocol. To demonstrate specificity of staining, the binding of PE-C15.6 antibody was blocked by the preincubation of the conjugated antibody with recombinant mouse IL-12 p40 protein (0.5 µg, Cat. No. 554594; see middle panel), and by preincubating the fixed/permeabilized cells with unlabeled C15.6 antibody (5 µg/ml final concentration; Cat. No. 554477; see right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control and verified using the ligand blocking and unlabeled antibody blocking controls.