Ficoll hypaque

Human whole blood (WB) was obtained from healthy volunteers as part of a lung cancer metabolism study according to a University of Kentucky IRB approved protocol (IRB# 43807). Monocytes were isolated from WB using the RosetteSep™ human monocyte enrichment cocktail kit (StemCell Technologies Inc., Cambridge). Each mL of WB was mixed and incubated for 20 min with 2 μL of 0.5 mM EDTA, and 50 μL of the RosetteSep™ monocyte negative selection cocktail. The WB was diluted 1:1 with phosphate-buffered saline (PBS) (Ca²⁺ and Mg²⁺ free) + 1× antibiotic-antimycotic (Thermo fisher) + 2 % endotoxin-free fetal bovine serum (FBS) and then added to a Ficoll-Hypaque (GE) gradient in SepMate™-50 tubes (StemCell Technologies Inc.), followed by centrifugation at 1,200 ×g for 20 min. The top layers were pooled and then spun for 10 min at 400 ×g. The resulting cell pellets were resuspended in the same 2 % FBS-PBS dilution buffer and washed twice via centrifugation at 600 ×g and 120 ×g for 10 minutes each. Finally, the cells were counted and seeded into 6-well plates at a density of 3 – 5 × 10⁶ cells per well in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 0.2 % glucose, 2 mM glutamine, 10 % FBS, 1× antibiotic-antimycotic, and 50 ng/mL of monocyte colony stimulating factor (M-CSF) (M0 medium).