Monocyte-Derived Macrophage Polarization Protocol

The study was approved by the local ethics committee (Bayerische Landesärztekammer, Munich) and subjects gave written, informed consent. Monocytes were enriched from whole blood by negative selection using the Rosette Sep® monocyte enrichment cocktail (STEMCELL Technologies) according to the manufacturer's instructions. Briefly, blood from healthy donors was collected in Falcon tubes containing EDTA at 2 mM final concentration and incubated with enrichment antibody cocktail (50 µl per ml of whole blood) at room temperature for 20 minutes. Cells were then separated by density gradient using Ficoll-Paque™ PLUS (GE Healthcare). Platelets present in the enriched monocyte fraction were discarded by 3 washing steps in PBS, 2% FBS. Finally, monocytes were seeded in either XVivo 10 (Cambrex) or RPMI 10% FBS, 4 mM L-Glutamine with Pen/Strep at a concentration of 5×10⁵ cells/ml in 12-well tissue culture treated plates for 6 days in the presence of either 100 ng/ml rHuGM-CSF (M1) or 100 ng/ml rHuM-CSF (M2). For M2a and M2c polarization, monocytes were incubated in XVivo 10 with rHuM-CSF and 10 ng/ml rHuIL-4 or 10 ng/ml rHuIL-10, respectively. For M1 activation, monocytes were first incubated with rHuM-CSF or rHuGM-CSF for 3 days followed by stimulation with 10 ng/ml LPS and 50 ng/ml rHuIFN-γ for 3 additional days. As indicated by the manufacturer, XVivo 10 media contains human albumin, recombinant human insulin, and human transferrin. It does not contain any exogenous growth factors, artificial stimulators of cellular proliferation, undefined supplements, or protein kinase C stimulators.
Changes in cell morphology were assessed by phase contrast microscopy (Axiovert 135, Zeiss). Phenotypical and functional characterization of MDM was performed after 6 days.