Human macrophage colony stimulating factor m csf

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.

Peripheral blood mononuclear cells were isolated from human blood after ficoll gradient separation (GE Healthcare) and CD14⁺ cells were isolated using CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Cell numbers were determined using a Neubauer chamber by Trypan blue staining, and cells were seeded at a density of 1 × 10⁵ cells per well in 96-well plates (Corning). Monocytes were differentiated into macrophage for 4 days in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific) and 50 ng/ml human macrophage colony-stimulating factor (M-CSF; Miltenyi Biotec). The growth medium was changed to RPMI supplemented with 10% FBS prior to infection. Macrophages were grown at 37 °C with 5% CO₂.