Isolation and Culture of Human Monocyte-Derived Macrophages

Mononuclear cells were separated by density gradient centrifugation [40 (link)]; whole blood was centrifuged at 300 g for 15 min, Buffy coats were diluted 1:1 in warmed Dulbecco’s phosphate-buffered saline (D-PBS; Sigma, UK) and over-layered onto Ficoll-Paque (Histopaque-1077; Sigma, UK), then centrifuged at 400 g for 30 min. Contaminating red cells were removed using erythrocyte lysis buffer (ELB) containing 0.15 M ammonium chloride [41 (link)]. Cells were then labelled with anti-human CD14 conjugated to magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions and passed over magnetic columns to isolate CD14⁺ cells [31 (link), 42 (link)]. Following this process, numbers of dead cells were negligible (determined by Trypan blue). Cells were suspended in RPMI with glutamine (Sigma UK) supplemented with 10% FCS (Sigma), containing 200 U/ml penicillin (Sigma) and 200 mg/ml streptomycin (Sigma) at 5–20 x 10⁵cells/ml (1–4 x 10⁵cells/well) in 96-well plates (Costar) in triplicate for biochemical assays or in 24-well plates (Costar) for RNA extraction (3–4 × 10⁶ cells/well). Cells were incubated at 37 °C in 5% CO₂, medium being replaced every 2 days, until cells achieved 80% confluence and they had matured into monocyte-derived macrophages (MDM), at 6–12 days culture.