Differentiation of CD14+ Monocytes into Macrophages

CD14⁺ monocytes were isolated from blood of human donors (approved by the local ethical committee, 2007–2019, with written consent obtained from each donor) by centrifugation through Ficoll-Paque (Amersham, GE Healthcare, Little Chalfont, UK), and subsequently suspended in 0.5% bovine serum albumin (BSA) and 2 mM EDTA in PBS, and were purified by using BD IMag™ Anti-Human CD14 Magnetic Particles - DM (BD Biosciences, CA) according to the supplier's instructions (Moller et al., 2017 (link)). CD14⁺ cells were seeded at a density of 66,667 cells/cm² in T75 culture flasks (Greiner, InVitro, Fredensborg, Denmark) supplied with α minimum essential medium (αMEM; Invitrogen, Taastrup, Denmark) containing 10% fetal calf serum (FCS; Sigma-Aldrich, St Louis, MO) and 25 ng/ml human macrophage colony-stimulating factor (M-CSF; R&D Systems, Abingdon, UK) and cultured for 2 days at 37°C in 5% CO₂ in a humidified atmosphere (Soe and Delaisse, 2010 (link)). Floating cells were harvested by centrifugation (500 g for 5 min) and returned to the respective flasks in fresh αMEM with 10% FCS, 25 ng/ml human M-CSF and 25 ng/ml human RANKL (also known as TNFSF11). The cells were cultured for an additional 7 days with medium changed twice.