Human macrophage colony stimulating factor

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.

Human mononuclear leukocytes were isolated from the blood of healthy donors or TB patients and monocytes were obtained by magnetic-activated cell sorting (MACS) of CD14⁺ cells using CD14 MicroBeads (Miltenyi Biotec). Monocytes were differentiated into Mφ for 4 days in Roswell Park Memorial Institute 1640 (RPMI) medium supplemented with 10% fetal bovine serum (FBS) and 50 ng/ml human macrophage colony-stimulating factor (Miltenyi Biotec) at a density of 1 × 10⁵ cells per well in 96-well plates (Corning). Before infecting primary Mφ with varying MOI (1 or 2) of Mtb, the growth media was changed to RPMI supplemented with 10% FBS. Mφ were grown at 37 °C with 5% CO₂.