Objective To investigate the most appropriate condition for activating Th17 cells in vitro for ensuring to detect Th17 cells accurately by flow cytometry. Methods Peripheral blood mononuclear cells (PBMC) were stimulated by PMA at different concentration (25, 75, 150, 250 ng/ml), simultaneously add in ionomycin for 1 hour and with monensin for another 3 hours. And then Th17 cells were detected by flow cytometry after 4 or 6 hours. The result was compared with that was stimulated with Leukocyte Activation Cocktail. Results Th17 cells could activated effectively with 25 ng/ml PMA plus ionomycin and monensin for the proportion of CD69+ T cells>90% and intracellular accumulations of IL-17. Leukocyte Activation Cocktail could activate Th17 cells to accumulate IL-17. But CD69 couldn’t be detected from the cell membrane. Conclusion The optimal condition for activating Th17 cells in vitro is PMA（25ng/ml）plus ionomycin（1μg/ml）for 1 hour and with monensin（1.7 μg/ml） incubating for another 3 hours.
