Untouched human t cell isolation kit

Peripheral blood mononuclear cells (PBMCs) were isolated by using Ficoll Histopaque gradient (GE Health Care Bio-Sciences, Piscataway Township, NJ). CD3⁺ T cells were isolated by negative selection using untouched human T cell isolation kit (Life technologies, Carlsbad, CA). Purity of CD3⁺ T cells was confirmed to be above 97% (data not shown). The cells were cultured in RPMI culture media (Corning CellGro, Manassas, VA) with 10% FCS (Gibco, Eugene, OR), 1% Penicillin/Streptomycin, and 1% L-glutamine (all from Corning CellGro) for 3 days either in the presence or absence of plate-bound anti-CD3 (anti TCR ε hybridoma from ATCC, Manassas, VA) and soluble anti-CD28 (BD Biosciences, San Jose, CA) with or without 100 nM rapamycin (Biotica, Cambridge, UK). In some experiments, IL-4 (100 ng/ml) (Peprotech, Rocky Hill, NJ, Catalog # 200-04), IL-17 (10 ng/ml) (eBioscience, San Diego, CA, Catalog# 14-8179), anti-IL-4 (5 µg/ml) (BioLegend, San Diego, CA, Catalog# 500815), or IL-17 (10 µg/ml) (R&D Systems, Minneapolis, MN, Catalog# MAB 317) was added to the culture media.