Pierce streptavidin plus ultralink resin

Microglial exosomes from the ischemic penumbra portion of the cortex were isolated as previously described [36 (link)]. In each group, the ischemic penumbra portion of the cortex was rapidly isolated from the left hemisphere. Samples were digested with collagenase type 3 (75 U/ml, Worthington Biochemical Corporation, Lakewood, NJ, USA) at 37 °C for 15 min. Then, the cells were centrifuged at 2000 × g at 4 °C for 10 min to remove dead cells and subsequently centrifuged again for 30 min at 10,000 × g at 4 °C to remove cellular debris. After collecting the supernatant, it was filtered through a 0.22 μm filter. Samples were centrifuged at 100,000 × g at 4 °C for 70 min, and the supernatant was removed. Calcium- and magnesium-free Dulbecco’s PBS (Gibco, USA) was applied to resuspend the exosome pellet. Samples were incubated for 1 h with 50 mL of 3% BSA containing 1.5 µg of anti-CD11b biotinylated antibody (NB110-89474B, Novus, CO, USA) at room temperature to isolate microglial exosomes. Subsequently, the mixture was incubated with Pierce Streptavidin Plus UltraLink Resin (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 30 min. Thereafter, centrifuged at 800 × g for 10 min at 4 °C of the samples was performed. The supernatant was discarded after centrifugation, and microglial exosomes were recovered and stored at 4 °C until use in subsequent analysis.

tEVs were isolated from serum using ExoQuick solution (System Bioscience, Palo Alto, CA, USA) with some modifications to the manufacturer’s instructions. After centrifugation to remove cell debris, the serum of six animals in each group were pooled. A total of 2 mL of serum and 500 μL of solution were mixed. After the mixture was centrifuged, the pellet was resuspended in 200 μL phosphate-buffered saline (PBS).
nEVs were isolated as described by Mustapic et al. [52 (link)] with some modifications. Exosomes isolated from serum were incubated with anti-CD171 (L1CAM; Bioss Antibodies, Beijing, China) antibody for 1 h at 4 °C on a rotating mixer. After adding Pierce Streptavidin Plus Ultralink Resin (Thermo Fisher Scientific, Waltham, USA) and PBS, the samples were again incubated for 1 h at 4 °C on a rotating mixer. The samples were then pelleted by centrifugation at 200× g for 10 min at 4 °C. The supernatants were removed from the samples and the pellets were resuspended in 200 μL 0.1-M glycine-HCl (Biosesang, Seongnam, Korea). After mixing for 10 s and vortexing for 30 s, the samples were pelleted by centrifugation at 4500× g for 10 min at 4 °C. Finally, the supernatants were transferred to new tubes before Tris-HCl (Biosesang) and PBS were added.

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.