PBMC isolation and macrophage differentiation

Peripheral blood mononuclear cells (PBMCs) were isolated as previously described.²¹ In brief, whole blood from healthy volunteers was separated using lympholyte‐poly (Cedarlane Laboratories) according to the manufacturer's instructions. Whole blood was obtained in compliance with the Office of Research Ethics at The University of Western Ontario (protocol 109059). Isolated mononuclear cells were allowed to adhere to 18-mm glass coverslips in 12-well tissue culture plates. After a 1-hour incubation, the cells were washed to remove nonadhered cells and then incubated in RPMI 1640 containing 10% (vol/vol) fetal bovine serum supplemented with penicillin-streptomycin antibiotic cell culture solution (Wisent Bioproducts) and recombinant human macrophage colony-stimulating factor (M‐CSF) at 10 ng/mL (PeproTech). Macrophages were differentiated until day 9 postisolation in medium containing 10 ng/mL recombinant human M-CSF, at which time phagocytosis assays were performed.