Isolation of Human Monocytes from Blood

Human monocytes were isolated from 500 ml fresh whole blood (drawn within 1 h before use) of healthy male donors. Blood was layered onto an equal volume of 1-Step Polymorphs (Accurate Chemical & Scientific Corporation, USA) and centrifuged at 650 × g for 35 min. After centrifugation, the peripheral blood mononuclear cells (PBMCs) were collected, and normal osmolarity was restored by adding an equal volume of 0.45% cold NaCl. After erythrocyte lysis using a hypotonic buffer, cells were washed twice in cold PBS and counted using a Neubauer chamber. Cell viability of >95% was assessed by trypan blue staining. Monocytes were isolated from the PBMCs using the monocyte isolation kit II and quadro-MACS (Miltenyi Biotec, UK), following manufacturer’s instructions. Purity of the monocytes (>90%) was assessed as described in Klassert et al.32 (link).