Isolation and Cryopreservation of Human PBMCs

Human PBMCs were isolated from buffy coats of healthy volunteers (obtained at Portuguese Institute of Blood and Transplant) by a Ficoll-Paque PLUS density gradient (GE Healthcare, UK). PBMCs were then resuspended in RPMI-1640 medium (Lonza, Switzerland), supplemented with 10% (v/v) heat-inactivated FBS (HyClone, USA), 2 mM of L-glutamine (Lonza, Switzerland), 1% (v/v) Pen/Strep/Fungiezone solution (HyClone, USA) and placed at 37 °C in a humidified atmosphere of 5% CO₂ overnight. After this recovering step, PBMCs were counted and frozen in freezing medium (10% DMSO, v/v, in FBS) and stored at −80 °C. When needed, PBMCs were thawed and cultured in the same supplemented-RPMI-1640 medium as before. PBCMs were maintained overnight at 37 °C in a humidified atmosphere of 5% CO₂, to decrease loss of effector capacity^{65 (link)} and only then used in the ADCC assays.