Primary monocyte isolation and culture

Leukopaks were obtained from the New York Blood Center and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque(GE Health care, Uppsala, Sweden). Monocytes were isolated from PBMCs using EasySep Human CD14 Positive Selection Kit according to the manufacturer's instructions (Stemcell Technologies, Vancouver, BC, Canada). CD14⁺ monocytes were cultured non adherently in teflon coated flasks at a density of 2 × 10⁶ cells/ml in RPMI 1640 media (Thermo Fisher Scientific) supplemented with 10% heat-inactivated human serum type AB and 5% heat-inactivated fetal bovine serum (Lonza, Walkersville, MD), 1% HEPES , 1% penicillin/streptomycin, and 10 ng/ml M-CSF (Peprotech) as previously described (44). This culture system yields 70% mature CD14⁺ CD16⁺ monocytes (44).