Differentiation of Primary Human Macrophages

Primary human macrophages were differentiated from monocytes, according to the protocol reported previously.⁴¹ (link) In brief, monocytes were purified from buffy coat-derived peripheral blood mononuclear cells (Deutsches Rotes Kreuz, Berlin, Germany; ethics vote EA2/018/16; Charité University Medicine Berlin, Berlin, Germany), by magnetic sorting using the Monocyte Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instruction. CD14-positive monocytes were subsequently cultured in very low endotoxin (VLE) RPMI 1640 (PAN-Biotech, Aidenbach, Germany), supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich), and 50 ng/mL human macrophage colony stimulating factor (Miltenyi Biotec) at 37°C in an atmosphere with 5 vol% CO₂ for 7 days, with medium change at day 3. Upon differentiation, macrophages were cultured in VLE RPMI, only supplemented with 10% (v/v) FBS for subsequent experiments.