Human macrophage colony stimulating factor

293FT cells (catalog number R700–07; Thermo), HEK-293 cells (ATCC® CRL-1573™), Vero cells and HeLa cells were maintained in Dulbecco modified Eagle medium (Fisher Scientific) supplemented with 10% fetal bovine serum (Atlanta Biologicals) (referred to here as complete medium). Primary human macrophages were differentiated from the buffy coat fraction of human blood (from the South Texas Blood and Tissue Center) according to previously published protocols.⁵ Briefly, mononuclear lymphocytes were isolated using LeucoSep tubes (Fisher Scientific), resuspended in Iscove modified Dulbecco medium (IMDM, Fisher Scientific), and plated in 96-well plates (50,000 cells per well). After the cells were allowed to adhere for 1 h, unattached cells were washed off using IMDM. Attached monocytes were allowed to differentiate into macrophages for 7 to 8 days in IMDM containing 2% heat-inactivated human serum (Corning Inc.), 100 U/ml penicillin, 100 μg/ml streptomycin, 1× nonessential amino acids (Fisher Scientific), 50 μM 2-mercaptoethanol (Fisher Scientific), and 800 U/ml human macrophage colony-stimulating factor (BioLegend). Adherent monocytes (PHMs) were washed, and the medium was replaced on days 2 and 6 while the cells were differentiating. All cells were kept at 37°C in a humidified incubator with 5% CO₂.