Kinetics of cytokine production by splenic iNKT cells. (A,B) C57BL/6 splenocytes were purified via a density-gradient and stimulated either for 0.5–4 h with PMA/ionomycin (A) or for 1–5 h with αGalCer (B) in the presence of the Golgi-transport inhibitors Brefeldin A and monensin. Expression of the indicated cytokines by splenic iNKT cells was analyzed by ICCS. A summary graph of the absolute values of cytokine positive iNKT cells over time is shown in the top panels. The lower panels show normalized data, with the relative increase of cytokine positive iNKT cells at the last time point (PMA/ionomycin = 4 h, αGalCer = 5 h) set to 100%. The graphs summarize the mean values of three independent experiments done in parallel for each stimulation method, with three mice in each experiment.