Isolation and Culture of Human Monocytes

Monocyte isolation via positive selection was performed according to the manufacturer’s protocol StraightFrom^® Whole Blood CD14 MicroBeads (human, Miltenyi Biotec, Bergisch Gladbach, Germany). The negative selection was performed using the EasySep^TM Direct Human Monocyte Isolation Kit (STEMCELL, Vancouver, BC, Canada) as previously described [22 (link)]. After the isolation, monocytes were diluted in RPMI 1640 medium (specification: very low endotoxin), supplemented with 2 mM stable glutamine, 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (all: PAN-Biotech, Aidenbach, Germany). The cells were seeded in cell-repellent multiwell plates with a concentration of 1 × 10⁶ cells/ml and incubated at 37 °C and 5% CO₂ according to the applied experimental design.