Isolation of Extracellular Vesicles from Macrophages

Extracellular vesicles were prepared as previously described (Skog et al. 2008 (link)) with total exosome isolation (from cell culture media) kit (Invitrogen). Briefly, human monocytes were cultured and differentiated for 7 d to become macrophages. Macrophages were treated with PM_2.5 in vesicle-free medium (Neurobasal medium), and conditioned medium from 1.1 × 10⁶ cells/well was collected after 24 h. Extracellular vesicles were purified by differential centrifugation. Macrophage-conditioned medium was centrifuged for 5 min at 300 g to remove cells. Supernatants were further centrifuged for 20 min at 4,000 g. Additional supernatants were mixed with half volume of total exosome isolation reagent and centrifuged at 10,000 g for 1 h. The extracellular vesicle pellets were resuspended in M-PER Protein Extraction Buffer (Pierce) for western blot or in neurobasal medium for HPLC after 48 h incubation with and without glutamine at 37°C.