Macrophage Differentiation and Exosome Response

Human monocytic cells U937 were differentiated towards macrophages following a previously published protocol [27 (link)]. Briefly, 5 × 10⁵ cells/mL were cultured in RPMI-1640 with 10% heat-inactivated FBS and 100 ng/mL PMA (phorbol-12 myristate 13-acetate, Sigma Aldrich, St. Louis, MO, USA) for 48 h, and then the culture medium was changed. After 24 h, the cells were gently scraped and treated with 100 ng/mL LPS (Sigma Aldrich, St. Louis, MO, USA) for 10 min. The cells (un-stimulated and stimulated) were then seeded in 24-well plates and incubated with exosomes or vehicle (control) for 18 h, after which the medium was collected and the TNFα concentration was quantified using a human TNFα Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA).