Papain

Fresh or previously frozen murine hemi-brains were dissected and treated with 20 units/ml papain (Worthington) in Hibernate E solution (3 ml/hemi-brain; BrainBits, Springfield, IL) for 15 min at 37 °C. The brain tissue was gently homogenized in 2 volumes (6 ml/hemi-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). Exosomes were isolated from the filtrate as described previously (15 ). Briefly, the filtrate was sequentially centrifuged at 300 × g for 10 min at 4 °C, 2000 × g for 10 min at 4 °C, and 10,000 × g for 30 min at 4 °C to discard cells, membranes, and debris. The supernatant was centrifuged at 100,000 × g for 70 min at 4 °C to pellet exosomes. The exosome pellet was resuspended in 60 ml of cold PBS (Invitrogen), and the exosome solution was centrifuged at 100,000 × g for 70 min at 4 °C. The washed exosome pellet was resuspended in 2 ml of 0.95 m sucrose solution and inserted inside a sucrose step gradient column (six 2-ml steps starting from 2.0 m sucrose up to 0.25 m sucrose in 0.35 m increments, with the 0.95 m sucrose step containing the exosomes). The sucrose step gradient was centrifuged at 200,000 × g for 16 h at 4 °C. One-ml fractions were collected from the top of the gradient, and fractions flanking the interphase separating two neighboring sucrose layers were pooled together for a total of seven fractions (a, top 1-ml fraction; b, 2-ml; c, 2-ml; d, 2-ml; e, 2-ml; f, 2-ml; and g, bottom 1-ml fraction). These fractions were diluted in cold PBS and centrifuged at 100,000 × g at 4 °C for 70 min. Sucrose gradient fraction pellets were resuspended in 20 μl of cold PBS. Two μl were used to measure acetylcholine esterase (AChE) activity, and 2-μl were used for EM. Exosome lysate was prepared by mixing 16 μl of the leftover solution with an equal volume of 2× radioimmune precipitation assay lysis buffer supplemented with a mixture of protease inhibitors. We used 2 μl of the lysate to quantify exosomal protein content (BCA protein assay kit, Pierce) and 10 μl of the lysate (31% of the exosome lysate total volume) for protein analysis by Western blotting.