Human peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll-Paque™ Plus; GE Healthcare) of buffy coat preparations from anonymous donors (Institute of Transfusion Medicine, Ulm University). Monocytes were isolated from PBMCs by adherence on tissue culture treated plastic flasks (Falcon, Corning, NY, United States). Monocyte-derived macrophages were generated by incubation with granulocyte-macrophage colony-stimulating factor (10 ng/ml; Miltenyi Biotec, Bergisch Gladbach, Germany) in Macrophage-Serum Free Medium (M-SFM; Gibco, ThermoFisher Scientific, Schwerte, Germany) for 6 days. Phenotypic characterization by flow cytometry demonstrated that macrophages expressed CD68 (anti–CD68-FITC, clone Y1/82A; BD Biosciences, Franklin Lakes, NJ, United States) and MHCII (anti–HLA-DR-PerCP, clone L243; BD Biosciences) as described (Bruns et al., 2012 (link)).
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