Isolation and Differentiation of Macrophages

Polymorphonuclear granulocytes (PMN) and peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of healthy donors by density gradient centrifugation using either Polymorphprep^® (Progen, Heidelberg, DE) or Ficoll Paque Plus (GE Healthcare, Chicago, IL, USA), respectively, as previously described (19 (link), 20 (link)). PBMC were then used for generation of non-polarized (M0) macrophages as described (17 (link)). Briefly, after incubation in monocyte attachment medium (PromoCell, Heidelberg, DE) for 30 min at 37°C, cells were washed three times with PBS to dispose non-adherent cells and resuspended in X-VIVO 15 medium (Lonza, Basel, CH) supplemented with 0.5% v/v penicillin/streptomycin (Gibco, Amarillo, TX, USA). After culturing for 24 h, 50 ng/ml M-CSF (PeproTech, Rocky Hill, CA, USA) were added and refreshed every 72 h at least twice before macrophages were used in ADCP experiments.