Isolation of Primary Human T-Cells

Frozen human peripheral blood mononuclear cells (hPBMC) were purchased from Lonza (Basel, Switzerland) and were initially collected from healthy donors under their IRB protocol. To minimize known inter-patient variability occurring across sex, age, and ancestry,[57 (link)] hPBMCs were purchased from healthy non-smoking male donors between the age of 35–50 years old of African American ancestry. The cells were thawed and incubated for 4 hours in media (RPMI-1640 medium; Corning, Tewksbury, MA) supplemented with 10% heat-inactivated fetal bovine serum (hiFBS) and 1% penicillin-streptomycin (P/S), defined as complete media, at 37 °C, 5% CO₂. After 4 hours, CD3+ primary T-cells were isolated using Human Pan T-Cell Isolation Kit (Miltenyi Biotec, Auburn, CA) following the manufacturer’s protocol. T-cell purity (>97%) was determined by staining the cells with anti-CD3-AF700 and quantified using a flow cytometer (FACSAria Fusion, BD Biosciences, San Diego, CA). Isolated T-cells were either used directly for experiments or frozen down in a recovery freezing medium (90% hiFBS and 10% DMSO) for later use.