C 28051

Glass-bottom 24 well plates (Mattek, P24G-1.0-13-F) destined to seed monocytes were coated with Poly-l-Lysine (PLL, Millipore-Sigma, P7280) under sterile conditions and according to manufacturer's instructions. Briefly, 150 μl of a 100 μg/ml PLL solution was added on top of each well using endotoxin-free pipette tips and incubated for 30 min at room temperature. Next, PLL was aspirated and washed three times with cell culture grade water (Hyclone, SH3052901). PLL-coated surfaces were left to dry at room temperature for 1 h in sterile conditions.
Control monocytes were purified from whole blood using the Miltenyi Biotec MACSxpress Neutrophil Isolation Kit according to the manufacturer's instructions (Miltenyi Biotec, #130-104-434) and residual red blood cells were lysed using MACSxpress Erythrocyte Depletion Kit (Miltenyi Biotec, #130-098-196). All donors were healthy and exempt of informed consent according to the institutional review board (IRB 2016-0934). Monocytes were resuspended in attachment media (Promocell, C-28051) and seeded at a density of 7.5·104 cell/cm2 on the PLL-coated glass-bottom wells. After 45 min, monocytes had attached and media was changed to RPMI (ThermoFisher, 11875-093) with 10% FBS and 1% penicillin/streptomycin.

M1 macrophages were differentiated from freshly obtained PBMC using M1 macrophage Generation Medium DXF according to the manufacturer’s protocol (PromoCell GmbH, Heidelberg, Germany). In brief, freshly isolated PBMCs were seeded out in an appropriate amount of Monocyte Attachment Medium (C-28051, PromoCell GmbH) in a density of 1 million/cm² and incubated for 1–1.5 hours at 5% CO₂, 37°C. Subsequently, the non-adherent cells were removed by three washing steps with warm monocyte attachment medium, the M1 macrophage Generation Medium DXF (C-28055, PromoCell GmbH) was added to the remaining adherent monocytes and the cells were incubated with refreshment of the M1 macrophage Generation Medium DXF every 3 days for 9 days at 5% CO₂ and 37°C. At day 10, the differentiated M1-Macrophages were checked by flow cytometry for correct differentiation, transfected as described below and used for the mRNA microarrays and ELISAs.