Derivation of Human Macrophages from Monocytes

Macrophages were derived from primary human monocytes as previously described [24 (link)]. Briefly, monocytes were purified from peripheral blood mononuclear cells (PBMC) isolated from buffy coats (Deutsche Rote Kreuz, Berlin; ethics vote EA2/018/16; Charité University Medicine Berlin) by negative selection using the Monocyte Isolation Kit II (Miltenyi Biotec, Germany) according to the manufacturer’s instruction. Purified cells were cultured in very low endotoxin (VLE) RPMI 1640 (Biochrom, Germany), supplemented with 10 vol% FBS (Sigma, Germany) and 50 ng mL⁻¹ human macrophage colony stimulating factor (M-CSF) (Miltenyi Biotec, Germany) at 37 °C and 5 vol% CO₂ for 6–7 days, with medium changes every third day. At the end of the differentiation period at day 7, the medium was replaced with warm VLE RPMI supplemented only with 10 vol% FBS.