Anti cd14 beads

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density gradient centrifugation using Pancoll human (PAN Biotech, Aidenbach, Bayern, Germany). To positively select for CD14⁺ monocytes, 200 μl of anti-CD14 beads (Miltenyi Biotec, Bergisch-Gladbach, Germany) were added to 5-10x10⁸ PBMCs for 20 min and cells were separated by an autoMACS classic (Miltenyi Biotec, Bergisch-Gladbach, Germany) using the program ‘Possel’. Macrophages were generated in vitro by culturing CD14⁺ monocytes in complete RPMI containing 10 ng/ml human M-CSF in 6-well cell culture plates at a cell concentration of 0.33 x 10⁶/ml and a density of 0.1x10⁶/cm² for 6 days, at 37°C and 5% CO_2.

Human peripheral blood samples were obtained from healthy donors at the University Clinic of Erlangen (Ethical Committee Erlangen approval number 111_12B). Peripheral blood mononuclear cells (PBMCs) were harvested from Leukoreduction system chambers using a Pancoll-gradient centrifugation protocol as described earlier (22 (link)). In brief, Leukoreduction system chamber content was diluted with PBS (Merck) and layered onto Pancoll (Pan Biotech) for density centrifugation (1,328 x g, 25 min, room temperature). The leukocytes were collected and the remaining erythrocytes lysed via a brief incubation in cold ddH₂O. After washing in PBS, PBMCs were positively selected using anti-CD14 beads (Miltenyi Biotec) according to the manufacturer’s protocol. The selected cells were cultivated for seven days in cRPMI (complete RPMI, RPMI 1640 containing 10% FCS, 1% HEPES, 0.5% 2-mercaptoethanol, 1% Penicillin/Streptomycin and 5µl/ml human M-CSF [≙ 50 U/ml, Peprotech]) in cell culture flasks. After seven days, cells were detached with Accutase/PBS (1:4) for 30 min at 37°C and gentle rinsing with PBS, before they were harvested by centrifugation (300 x g, 5 min, room temperature), resuspended in cRPMI w/o antibiotics, and seeded into cell culture plates. The cells were incubated at 37°C and 5% CO₂ until further processing.