Th17 Cell Induction from Naïve CD4+ T Cells

The polarized Th0 and Th17 cells from naïve CD4⁺ T cells were rinsed with PBS re-cultured in RPMI 1640 medium containing 2 mM glutamine and 10% FBS in the presence of 50 ng/mL phorbol 12-myristate 13-acetated (PMA) and 1 μM ionomycin for 6 hours. 1 μg/mL brefeldin A was added for the last 3 hours of culture (all purchased from Sigma Aldrich). Thereafter, cells were stained for surface markers (anti-CD4, GK1.5, BioLegend). Intracellular cytokine staining (anti-IL-17A, TC11–18H10.1, BioLegend) was performed as described previously (28 (link)). Approximately 100,000 cells were analyzed on a BD FACSCanto II flow cytometer (BD Biosciences). The Th17 population was defined as CD4⁺IL-17A⁺.