Differentiation of THP-1 Monocytes to Macrophages

THP-1 monocytes were obtained from ATCC® (Manassas, VA, USA). Cells were maintained in RPMI 1640 medium (ATCC®) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (GIBCO®, Waltham, Massachusetts, USA) and 0.05 mM 2-mercaptoethanol (Sigma®, St. Louis Missouri, USA) during 5 days in a humidified incubator at 37°C with 5% of CO₂. THP-1 monocytes were differentiated to macrophage-like cells as previously reported [23 (link)]. Briefly, cells were exposed to 50 ng/mL phorbol myristate acetate (PMA) (Sigma®) during 24 h and then incubated for additional 24 h in the absence of PMA before further treatment. Macrophage-like THP1 cells were harvested by washing them with cold PBS (Gibco®) with 1% FBS and then exposed to cold 25 mM EDTA (Invitrogen®, AM9260G) for 10 min in ice. Cells were gently removed, suspended, and washed with RPMI 1640 medium containing 10% FBS. Cells were counted, and the viability was assessed using trypan blue (Sigma®, T8154-100ML). In all experiments, we used microscopy to confirm that PMA treatment induced the expected morphological changes; we also evaluated CD14 and CD11 expression by flow cytometry using anti-CD14 (BD Biosciences, La Jolla, CA, USA) and anti-CD11b (BioLegend®, San Diego, CA, USA) antibodies.