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, Cat. No. 554586). Adherent cells were washed and treated for ~14 hr with mouse IFN-γ (10 ng/ml, 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 hr. 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 surface stained with 0.06 µg of FITC-conjugated rat anti-mouse CD11b (MAC-1) antibody (Cat. No. 553310; see left panel). Cells were then fixed, permeabilized, and nonspecific binding to intracellular antigens was blocked using PBS/2%BSA/0.1% saponin. Cells were then stained with 0.25 µg of APC-conjugated rat anti-mouse IL-12 (p40/p70) antibody (APC-C15.6, Cat. No. 554480; see middle panel) using the staining protocol. To demonstrate specificity of staining, the binding of APC-C15.6 antibody was blocked by the preincubation of cells with unlabeled C15.6 antibody (5.0 µg/ml, Cat. No. 554477; see right panel) prior to staining with APC-C15.6. 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. This APC-conjugated reagent can be used in any flow cytometer equipped with a a dye, HeNE or red diode laser.  These include the dual laser FACStarPLUS™, FACS Vantage™ or FACSCalibur™.