Astrocyte-Derived Exosome Isolation

DPBS-washed astrocytes were seeded into new culture flasks (107 cells/flask) and cultured in a medium containing FBS without exosomes (SBI System Biosciences, Palo Alto, CA, USA). Some 12 h later, the cells were stimulated with a cocktail of proinflammatory cytokines: IL-1a (10 ng/mL, R&D Systems, Ixonia, WI, USA), TNF-α (30 ng/mL, R&D Systems), C1q (400 ng/mL, R&D Systems). Alternative phenotypes of astrocytes were induced by IL-10 (10 ng/mL) or TGF-β (10 ng/mL) stimulation (both cytokines from R&D Systems). Astrocytes cultured without cytokines were also included. In our previous work, we characterized the secretory activity of astrocytes in response to these stimuli [81 (link)]. After a six-day culture, the media were collected and centrifuged (300× g, 10 min, 20 °C) and the supernatants were centrifuged again (16,000× g, 30 min, 20 °C). Next, the exosomes were precipitated (12 h, 4 °C) with exosome precipitation reagent (Exo-spin Buffer, CellGS, Cambridge, UK) and centrifuged (16,000× g, 1.5 h, 20 °C). The supernatants were removed and the pellets were resuspended in PBS and purified on exosome size-exclusion columns (Exo-spin, Cell Guidence Systems, Cambridge, UK) according to a protocol provided by the assay manufacturer. After purification, the exosomes were aliquoted and frozen for further procedures.