Isolation and Characterization of Human Monocytes

Freshly drawn human whole blood anticoagulated with EDTA was diluted 1:2 in PBS containing 5 mM EDTA, and peripheral blood mononuclear cells (PBMCs) were enriched by density gradient centrifugation on Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden) as previously described^{52 (link)}. Monocytes were isolated from PBMCs by negative depletion of non-monocytes (Pan Monocyte Isolation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany) as previously described^{42 (link)}. Their purity was determined by cell counting (Sysmex KX-21N, Sysmex, Neumuenster, Germany) as well as by flow cytometry after staining with anti-CD45-PB and anti-CD14-PE as monocyte markers as well as anti-CD41-PC7 for platelet origin. Monocyte purity was calculated as ratio of CD14⁺CD45⁺ cells (monocytes) to all CD45⁺ cells. Monocyte viability was assessed by trypan blue exclusion. Monocytes were kept in RPMI medium supplemented with 10% EV-depleted AB serum in suspension culture plates (Greiner Bio-One) overnight prior to further use.