Cell culture

Pfeiffer cells, originally from the American Type Culture Collection, were cultured in RPMI 1640 (Gibco, USA) containing 10% FBS and 1% penicillin/streptomycin maintained at 37°C in a humidified 5% CO₂ incubator. Cells were passaged every three days. PBMCs were separated from the uncoagulated blood by a density gradient centrifugation method using Ficoll Histopaque in a 15 mL centrifuge tube that was centrifuged for 20 min at 750×g in a swinging bucket without braking. The PBMCs in the interphase were aspirated and washed twice with sterile PBS by centrifugation at 300×g for 10 min. The pellet was resuspended by complete medium and cultured like the Pfeiffer cells.