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).