Isolation and Differentiation of Human Monocyte-Derived Macrophages

Human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Paque (GE healthcare) density centrifugation from whole blood donated by healthy volunteers. The South Sheffield Research Ethics Committee approved the studies, and subjects gave written, informed consent. Monocytes were enriched from freshly isolated PBMC using MACS Monocyte Isolation Kit II and MACS LS Columns (Miltenyi Biotec), yielding an average 98% purity. To differentiatiate PBMC into monocyte-derived macrophages (MDM) 2×10⁶ PBMC/mL were plated in RPMI 1640 media (Lonza) with 2 mmol/L L-glutamine (Gibco BRL) containing 10% human AB serum (First Link (UK) LTD) in 24-well plates (Costar). After 24 h, non-adherent cells were removed, and adherent cells were cultured in RPMI with 10% heat-treated fetal bovine serum (FBS; Bioclear) in 5% CO₂ at 37°C to give a final concentration of approximately 2×10⁵ MDM/ml at 14d [17] (link). The THP1 cell line was obtained from ATCC, and maintained at 2×10⁵ cells/ml in RPMI 1640 medium supplemented with 10% FCS and 2 mmol/L L-glutamine. THP1 cells (2×10⁵/ml) were differentiated using 100 nM Vitamin D₃ (VD₃, Sigma-Aldrich) or 200 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) for 3d. Differentiation of PMA treated cells was enhanced after the initial 3d stimulus by removing the PMA-containing media then incubating the cells in fresh RPMI 1640 (10% FCS, 1% L-glutamine) for a further 5d (PMAr).