Isolation and Culture of Human Monocyte-Derived Macrophages

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood of healthy volunteers by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich). The cells were washed three times with sterile PBS and resuspended in RPMI 1640 (Life Technologies) supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin (complete medium). The PBMCs were incubated at 37 °C in complete medium and allowed to adhere for 45 min. The nonadherent cells were removed and the adherent cells were washed with sterile PBS and harvested with a rubber policeman. The purity of the monocytes isolated by Ficoll-Hypaque as determined by flow cytometry was approximately 85–90%. The cells were cultured for three weeks in conditioned mediums from THLE-2 cells in the presence of 25 ng mL⁻¹ recombinant human M-CSF (R&D Systems). The medium was changed every other day. On day 21, TRAP-positive cells were identified using a leukocyte acid phosphatase kit (Sigma-Aldrich)^{10 (link)}.