Culturing Memory CD4+ T Cells and Th17 Polarization

RPMI 1640 medium and fetal bovine serum for culturing memory CD4⁺ T cells and preparing polarized Th17 cells were purchased from Hyclone. Human T-cell activator anti-CD3 and anti-CD28 antibody cocktail covalently bound to magnetic beads (Dynabeads) was obtained from ThermoFisher Scientific. Soluble anti-CD3 and anti-CD28 antibodies were from BD Biosciences. Ingenol-3-angelate and SAHA were purchased from Cayman Chemical. Recombinant TNF-α was from R&D Systems. Prostratin, PMA and ionomycin were from Millipore Sigma. The sources and catalog information of all commercially obtained inhibitors and antibodies used in the current study are shown in S1 Table. HEXIM1 antibody was custom-synthesized and affinity purified by Covance Research Products. Generation, purification and validation of the polyclonal antibody towards the phospho-Ser-175 CDK9 epitope has been described previously [39 (link)].