Hibernate e solution

Exosomes were isolated from brain extracellular spaces as previously described (14 (link)). Fresh rat brains were dissected and treated with 20 units/ml papain (Worthington) in Hibernate E solution (6 ml/brain; BrainBits, Springfield, IL) for 15 min at 37°C. The brain tissue was gently homogenized in 2 volumes (12 ml/brain) of cold Hibernate E solution. The brain homogenate was sequentially filtered through a 40 μm mesh filter (BD Biosciences) and a 0.2 μm syringe filter (Thermo Scientific). The filtrate was centrifuged at 300× g for 10 min, then at 3,000× g for 20 min, and 10,000× g for 30 min. Exosomes were collected through ultracentrifugation at 100,000× g for 2 h. All centrifugation steps were performed at 4°C.

EV isolations from the brains were carried out as described previously with modifications according to the protocol [22 (link)]. The fresh and previously frozen mice hemibrains were harvested and dissected finely. The brain samples were then treated with 20 units/ml papain (Worthington) in Hibernate E solution (BrainBits, Springfield, IL) for 15 min at 37 °C. The same volume of cold Hibernate E solution was added to the brain samples to stop the reaction of papain. The brain tissue was then gently homogenized and filtered through a 40-μm mesh filter (BD Biosciences), followed by a centrifugation at 300×g for 10 min and 3000×g for 20 min at 4 °C to get rid of cells, membranes, and debris. After the supernatants were filtered through 0.45-μm filter (Thermo Scientific), they were subjected to 10, 000×g for 30 min at 4 °C to eliminate organelle contaminations. The supernatants were further centrifuged at 100,000×g for 70 min at 4 °C to pellet EVs. The pellets were then resuspended in filtered PBS, or MPER lysate solution for NanoSight or Western blot. All the samples were ultracentrifuged in ultraclear polycarbonate tubes (Beckman Coulter) that have a volume of 13.2 ml. A Beckman Coulter ultracentrifuge (Beckman Coulter OptimaL-90K ultracentrifuge; Beckman Coulter, Fullerton, CA, USA) was used with a rotor type SW 41 Ti.

Fresh frozen peri-ischemic striatum was dissected and treated with 20 units/ml papain (Worthington) in Hibernate E solution (2 ml/sample, BrainBits, Springfield, IL) for 15 min at 37°C. The tissue gently homogenized in 2 volumes (5 ml/sample) of cold Hibernate E solution. The tissue homogenate sequentially filtered through a 40 m mesh filter (BD Biosciences) and a 0.2 m syringe filter (Thermo Scientific). Exosomes were isolated from the filtrate as previously literature (Perez-Gonzalez et al., 2012 (link)). The filtrate was sequentially centrifuged at 300 g for 10 min at 4°C, 2,000 g for 10 min at 4°C, and 10,000 g for 30 min at 4°C to discard cells, membranes, and debris. Then, the exosomes were extracted according to the Exosome Isolation Kit (UR52121, Umibio, Shanghai, China). The total exosome RNA was isolated by the Exosome RNA isolation kit (Invitrogen), and microRNA expression profiling analyzed by the miRCURY LNA^TM microRNA array system (Exiqon).