Isolation and Culture of Human Macrophages

Human peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll-Paque Plus; GE Healthcare, Chicago, IL, USA) of buffy coat preparations from anonymous donors (Institute of Transfusion Medicine, Ulm University). Monocytes were selected from PBMCs by adherence on plastic. Monocyte-derived macrophages (referred to as “macrophages”) were generated by treatment with GM-CSF (10 ng/mL; Miltenyi Biotec, Bergisch Gladbach, Germany) in RPMI 1640 (Life Technologies, Carlsbad, CA, USA) cell culture medium supplemented with L-glutamine (2 mM; PAN Biotech, Aidenbach, Germany), HEPES (10 mM; Biochrom, Berlin, Germany), Penicillin/Streptomycin (100 µg/mL; Biochrom, Berlin, Germany) and 5% heat-inactivated human serum (Lonza, Basel, Switzerland) for 6 d. After culture, macrophages were detached with EDTA (1 mM; Sigma-Aldrich, Steinheim, Germany). Phenotypic characterization by flow cytometry demonstrated that macrophages expressed CD68 (anti–CD68-FITC, clone Y1/82A; BD Biosciences, Franklin Lakes, NJ, USA) and MHCII (anti–HLA-DR-PerCP, clone L243; BD Biosciences, Franklin Lakes, NJ, USA) as described [53 (link)].