P-FPMI-143 - P-FPMI-143
Venous blood (100 ml) from 5 healthy volunteers was collected in Vacutainer collection tubes containing sodium heparin as an anticoagulant (Becton Dickinson, Mississauga, ON). Blood was diluted 1:1 with complete RPMI 1640 medium (Gibco, Invitrogen Life technologies, Burlington, ON), supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS), 2 mM L-glutamine and 1 mM sodium pyruvate (all from Invitrogen Life Technologies), then separated by centrifugation over a Ficoll-Paque Plus (Amersham Biosciences, Piscataway, NJ, USA) density gradient. White blood cells were isolated from the buffy coat, washed twice in RPMI 1640 complete medium, and the number of peripheral blood mononuclear cells (PBMC) was determined by trypan blue exclusion. PBMC (2 x 10^7) were seeded into 6-well tissue culture dishes (Falcon; Becton Dickinson) at 5 x 10^6 cells/ml and incubated at 37 degree Celsius in 5% CO2 for 1 hr prior to activation.