Hibernate a solution

Brain EVs were isolated and purified as we have previously described.⁹ Briefly, frozen brain tissues were treated with 20 units/mL of papain (Worthington, Lakewood, NJ, USA) in Hibernate A solution (HA, 3.5 mL/sample; BrainBits, Springfield, IL, USA) for 15 minutes at 37°C. The brain tissue was gently dissociated in 6.5 mL of cold HA supplemented with protease inhibitors, centrifuged at 300 g for 10 minutes at 4°C to discard the cells, and the supernatant was sequentially filtered through a 40 μm mesh filter (BD Biosciences, San Jose, CA, USA) and a 0.2 μm syringe filter (Corning Life Sciences, Teterboro, NJ, USA). The filtrates were sequentially centrifuged at 4°C, at 2000 g for 10 minutes and 10 000 g for 30 minutes to discard membranes and debris, and at 100 000 g for 70 minutes to pellet the EVs. The EV pellet was resuspended in 60 mL of cold PBS (Thermo Fisher Scientific), and centrifuged at 100 000 g for 70 minutes at 4°C. The washed EV pellet was resuspended in 2 mL of 0.95 M sucrose solution and inserted inside a sucrose step gradient column (six 2‐mL steps from 2.0 to 0.25 M sucrose). The sucrose step gradient was centrifuged at 200 000 g for 16 hours and fractions were collected from the top of the gradient. The fractions were diluted in cold PBS and centrifuged at 100 000 g for 70 minutes for pellet collection.