Anti human cd14 conjugated magnetic beads

Human peripheral blood was obtained from healthy donors with approval of the local Ethics Committee (Charité-Universitätsmedizin Berlin) and were performed according to the Helsinki Declaration (ethical approval EA1/207/17, approved October 2017 and EA1/367/14, approved January 2015). Peripheral blood mononuclear cells from buffy coats were isolated by density gradient centrifugation using Ficoll-Paque^TM Plus technique (GE Healthcare Europe GmbH, Freiburg, Germany). CD14+ monocytes were enriched up to 99% purity and >95% viability (as verified by flow cytometry) by MACS using anti-human CD14 conjugated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany).
Monocytes were cultured at 1 × 10⁷ cells/ml in αMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1% Penicillin/Streptomycin (Thermo Fisher Scientific, Waltham, MA, USA), 100 ng/ml RANKL (PeproTech, Rocky Hill, NJ, USA) and 50 ng/mL M-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany) and varying doses of tofacitinib (0, 10, 100 nM; Sigma-Aldrich Chemie Gmbh, Munich, Germany) (Figure 6B). 100 µL of cell suspension was incubated under normoxic and hypoxic conditions in 96-well flat bottom LUMOX^TM plates (Sarstedt, Nümbrecht, Germany). Fully supplemented αMEM was refreshed every three days for 3–4 weeks and differentiation was monitored by microscopy.

Human peripheral blood was obtained from healthy donors. Peripheral blood mononuclear cells (PBMCs) were immediately isolated by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare). CD14⁺ monocytes were isolated by using anti-human CD14 conjugated magnetic beads (Miltenyi Biotec). To generate monocyte-derived macrophages, human CD14⁺ monocytes (2 × 10⁶ cells/mL) were cultured in culture medium (RPMI-1640 containing 10% FBS) supplemented with 100 ng/mL CSF-1 for 6 days. In the proliferation assay, differentiated macrophages (2×10⁴ per well) were incubated with serial dilutions of antibodies in culture medium in 96-well plates. CSF-1 (10 ng/mL) was then added to the cells. Cell survival was measured 72 h after stimulation using a CellTiter-Glo assay (Promega).

Monocytes were isolated from heparinized peripheral blood of healthy volunteers after giving written informed consent (ethical approval EA1/207/17 Charité - Universitätsmedizin Berlin). In brief, peripheral blood mononuclear cells (PBMC) from heparinized peripheral blood were isolated by density gradient centrifugation using Ficoll-Paque™ Plus (GE Healthcare, Chicago, USA). CD14⁺ monocytes were enriched from PBMC with >95% purity and >95% viability (data not shown, gating strategy provided in Figure S1) by using anti-human CD14 conjugated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and then immediately used for the experiments. Monocytes were cultured at 37°C in a humidified and atmosphere with 5% CO₂ (Binder, Tuttlingen, Germany) in aliquots of 300 µL at a density of 1x10⁷ cells/ml in 13 mL round bottom polypropylene tubes (Sarstedt, Nümbrecht, Germany) under orbital shaking at 120 min^-1 (KS250basic, IKA-Labortechnik, Staufen, Germany) in glucose free RPMI 1640 (Thermofisher, Waltham, USA) supplemented with 10% (v/v) dialyzed (glucose-free) human AB serum (Sigma-Aldrich, St. Louis, USA).