Isolation of Microglial Exosomes from Mouse Brain

The mice were sacrificed at 2, 5, 8 weeks after IH by transcardiac perfusion with PBS. Then, the hippocampus and cortex were detached to extract microglial exosomes [18 (link)]. In Brief, the tissue was digested with papain (Solarbio, Beijing, China) and then centrifuged at 4 °C for 10 min at 2000 g to remove cell debris. Further removal of cell particles was performed by centrifugation for 30 min at 10,000 g at 4 °C. Next, the supernatant was filtered through a 0.22 μm filter to remove dead cells and large particles.
After ultracentrifugation for 120 min at 100,000 g at 4 °C, the supernatant was removed, and the pellets were re-suspended in 350ul calcium- and magne-sium-free Dulbecco’s PBS (Thermo Fisher Scientific) and incubated for 60 min at room temperature with 1.5 mg rat anti-mouse CD11b bitinylated antibody (Thermo Fisher Scientific) in 50 µL of 3% BSA, followed by addition of 10 µl of Pierce Streptavidin Plus UltraLink Resin (Thermo Fisher Scientific) in 40 µL of 3% BSA and incubation for 30 min at room temperature with mixing. After centrifugation for 10 min at 800 g at 4℃, the supernatant was removed, and the pellets was suspended in 100µL of cold 0.05 M glycine- HCl (pH 3.0), mixed for 10 s [18 (link)]. After centrifugation for 10 min at 4000 g at 4℃, the supernatant was retained and stored for a short time at 4 ℃ for the next experiment.