Isolation and Differentiation of Human Monocytes

Human blood samples were collected into vacutainer EDTA tubes (Greiner Bio-One, Mosonmagyarovar, Hungary). PBMCs (peripheral blood mononuclear cells) were isolated via a Ficoll density gradient (Sigma, St. Louis, MO, USA). CD14+ cells were isolated from PBMCs using a positive magnetic selection method (EasySep, StemCell Technologies, Vancouver, Canada) (29 (link)). After isolation, a third of the monocytes were immediately frozen in liquid nitrogen. Two-thirds of monocytes were cultured (1x10⁵ monocytes per well) in MEM α (Minimum Essential Medium α) (Sigma, St. Louis, MO, USA) with 10% FBS (Gibco Laboratories, Gaithersburg, MD, USA), 1% L-glutamine (Sigma, St. Louis, MO, USA), and 1% Penicillin–Streptomycin (Sigma, St. Louis, MO, USA) at 37°C and 5% CO₂ in a humidified atmosphere. Monocytes were incubated with 50 ng/mL rh M-CSF (macrophage colony-stimulating factor) for 24 h (PeproTech, London, UK). Thereafter the samples were stimulated with 50 ng/mL of both rh M-CSF and rh RANKL (receptor activator of nuclear factor κB ligand) (PeproTech, London, UK). From then the media was replaced every 3-4 days.