P-FPMI-192 - P-FPMI-192
Venous blood (20 ml) from healthy volunteers was collected in Vacutainer® collection tubes containing sodium heparin as an anticoagulant (Becton Dickinson, Mississauga, ON) in accordance with UBC ethical approval and guidelines. Blood was diluted 1:1 with complete RPMI 1640 medium and 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 at 1x106 cells/ml were seeded into 6-well tissue culture dishes (Falcon; Becton Dickinson) at 37°C in 5% CO2. Subsequent to stimulation, CD14+ human monocytes were isolated from PBMC by direct positive selection by magnetic separation using Dynabeads CD14 coated with anti-CD14 monoclonal antibody (Dynal Invitrogen Life Technologies) as per the manufacturer’s instructions.
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