Cytokine and surface marker staining.
To assess cytokine production, cells were restimulated with 10 nM PMA + 1 μM ionomycin for 6 h (unless indicated otherwise in the figures), and intracellular cytokine stains were performed as previously described (28). To detect expression of surface molecules, cells were washed in PBS, resuspended in FACS wash buffer (3% FBS, 0.1% sodium azide, 30 mM Hepes, 1× PBS) containing the antibodies indicated in the figures at previously optimized concentrations, incubated for 15 min at room temperature (RT), washed, and resuspended in 2% formaldehyde fixative solution before acquisition on a FACSCalibur (BD).