Macrosep advance

Microglial medium was harvested, desalted, and concentrated by a Macrosep Advance centrifugal device with a molecular weight cutoff of 1 kDa (Pall Life Sciences, MI) to prepare cell supernatant samples. Microglial exosomes were isolated and purified by using an exosome isolation kit as described above. The protein concentration was determined by the Bradford protein assay. Proteins isolated from cell supernatants, whole-cell lysates and prepared exosomes were separated by SDS-PAGE (Millipore, Billerica, MA and Bio-Rad, Hercules, CA), the membranes were incubated overnight at 4 °C with primary antibodies. After washing, the membranes were incubated with horseradish peroxidase-linked anti-rabbit or anti-mouse secondary antibodies (1:20000, Jackson ImmunoResearch, USA) for 1.5 h at room temperature. The bands were visualised using chemiluminescence (BIO-RAD ChemiDoc MP Imaging System), and the density of each band was normalised to that of the loading control band, which was showed by silver staining using a ProteoSilver Silver Stain Kit (Sigma-Aldrich, USA). The loading control band was used to unify the loading quantity of exosome samples^{65 (link)}. All blots were processed in parallel and derive from the same experiment.

The supernatants of primary microglia collected from each sample were centrifuged at 2000 × g for 30 min to remove cell debris and dead cells (Beckman Coulter, Allegra X-14R). Exosomes were then purified from the supernatants using an exosome isolation kit (Thermo Fisher, UK, Cat^# 4478359). We added 0.5 volumes of the Total Exosome Isolation reagent into the required volume of cell-free culture media and then mixed the culture media/reagent mixture well by vortexing. After incubation at 4 °C overnight, samples were centrifuged at 10,000 × g for 1.5 h at 4 °C. The supernatant was aspirated and concentrated by a Macrosep Advance centrifugal device with a molecular weight cutoff of 1 kDa (Pall Life Sciences, MI) to prepare the exosome-depleted fraction, and the pellet containing exosomes at the bottom of the tube was resuspended in 150 μl RIPA buffer (Beyotime Biotechnology, China) to prepare the exosome-enriched fraction.