Preparation of peripheral blood mononuclear cells (PBMCs)
Buffy coat fraction was separated from blood samples by centrifugation (1,700 × g, 15 min, 25°C, without break). Next, PBMCs were purified from the buffy coat fraction by density gradient centrifugation (1,200 × g, 20 min, 25°C, without break) on 60% Percoll (GE Healthcare, Little Chalfont, UK). Then, collected PBMCs were washed 3 times by centrifugation (770 × g, 10 min, 25°C) in phosphate buffered saline and filtered through a 40-μm cell strainer (BD Biosciences, San Jose, CA, USA). Then, PBMCs were stained with 0.4% Trypan Blue Stain (Thermo Fisher Scientific, Waltham, MA, USA) and the number of the viable cells was counted using Countess II FL Automated Cell Counter (Thermo Fisher Scientific).