Primary human monocyte isolation and differentiation

Freshly prepared buffy coats were collected from healthy donors (Sylvan N. Goldman Oklahoma Blood Institute, Oklahoma City, OK, USA) by density gradient centrifugation as described previously^19,20. Briefly, PMBCs were purified using Ficoll Paque^TM (Fisher Scientific, Pittsburgh, PA, USA) based density centrifugation. PBMCs were incubated with magnetic labeled CD14 beads (Miltenyi Biotech, San Diego, CA, USA) according to manufacturer’s instructions. The purity of CD14+ cells was >95% as determined by flow cytometry. For MΦ differentiation, monocytes were plated at 2 × 10⁶/ml in DMEM supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml). After 2 h the media was substituted with media containing 10% FBS (Life Technologies, Carlsbad, CA, USA), and rhM-CSF (50 ng/mL; Peprotech, Rocky Hill, NJ, USA). For DCs, monocytes were cultured in RPMI-1640 supplemented with rhGM-CSF (1000 U/ml) and rhIL-4 (500 U/ml) (both from Peprotech). Media was replaced every 48 h. At day 7, cells were harvested and differentiation confirmed by flow cytometric analysis as described earlier^19,20.