PBMC Isolation and Overnight Culture

The study involved 15 shipments with 2-6 blood samples per shipment. Forty milliliter of blood was divided as follows: 8 mL for DNA extraction (PAXgene^®, Qiagen, Germantown, MD, USA, a BD company) and the remaining 32 mL into citrated tubes. The PBMCs were cultured overnight without stimulation [39 (link)] at 1–2 × 10⁶ cells/mL in 90% Dulbecco’ s complete media containing high (4.5 g/L) glucose and L-glutamine (Corning), 10% fetal calf serum (Atlanta Biologicals, Atlanta, GA, USA), 10 mM hepes (Sigma-Aldrich, St. Louis, MO, USA), and 1% penicillin-streptomycin (Sigma-Aldrich). Culture conditions and assay media were standardized with a single lot of fetal calf serum and one lot of tissue culture flasks (Biolite, Thermo Scientific, Waltham, MA, USA) throughout the study.