Ultracentrifuge

After fibrillization, 100 μL samples were centrifuged at 100,000 x g for 30 min at 4°C (Beckman Coulter ultracentrifuge). Pellets were resuspended in the same volume of PBS. The samples were then digested by adding either 2 μg/mL or 20 μg/mL of PK in PBS for 1 hour at 37°C. The reaction was stopped with 2 mM PMSF and the PK-digested samples were centrifuged at 100,000 x g for 1 hour at 4°C (Beckman Coulter ultracentrifuge). Pellets were resuspended in 1X SDS-PAGE loading buffer. The non-PK digested samples of fibril solutions were added into 2X SDS-PAGE loading buffer in a 1:1 ratio. The samples were boiled for 10 min at 100°C, loaded onto a 12% Tris-Glycine SDS- PAGE gel, and transferred overnight onto Immobilon P PVDF membranes (Millipore). Membranes were blocked by 5% non-fat milk, incubated first with 1 μg/mL anti-PrP Fab D18 and then with goat anti-human IgG F(ab)2 fragment HRP-conjugated. Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences).

Additional 3-month-old mice (n = 3–4) were CCI-induced or sham-induced. The TBI and sham mice were sacrificed after one day. The ipsilateral and contralateral sides of the brains were extracted from injured and sham mice. Brain hemispheres were homogenized at 10% w/v in PBS containing protease inhibitors (cOmplete™ Protease Inhibitor Cocktail, MilliporeSigma, Bedford, MA) as described previously.^{54 (link),55 (link)}Brain homogenates were centrifuged at 32,600 rpm for 1 h at 4°C in an ultracentrifuge (Beckman-Coulter). The supernatant was removed, and pellets were resuspended in 200 μL of 70% formic acid followed by sonication. Samples were centrifuged for 30 min under the same conditions, and the supernatant was collected and neutralized in 1 M Tris-HCl buffer, pH 10.8 to measure the fraction of insoluble tau in brain after TBI. Levels of phosphorylated tau at Ser199 were measured using Human tau [pS199] ELISA kit (Invitrogen) per manufacturer's instructions on an ELISA plate reader (EL800 BIOTEK).

Transmission Electron Microscopy (TEM) and Cryo-TEM analyses were performed using exosomes purified from cell culture supernatants by ultracentrifugation protocol [28 (link)]. Culture supernatants were centrifuged at 1000× g at room temperature for 10 min. The supernatants were collected and spun again at 10,000× g at room temperature for 30 min to remove cellular debris. The supernatants were filtered through 0.22 μm filters (Sigma-Aldrich, St. Louis, MO, USA), and exosomes were precipitated by ultracentrifugation at 100,000× g at room temperature for 2 h (Beckman Ultracentrifuge). The exosome pellet was resuspended in PBS for downstream analysis. Cryo-TEM was performed to demonstrate the purity and size of exosomes released from the HCV-infected cell culture using an FEI G2 F30 Tecnai TEM operating at 150 kV. The exosome samples were prepared on a lacey carbon-coated copper grid (200-mesh, electron microscopy sciences) using an automated plunging station (FEI Vitrobot). The sample solution was applied to the grid. The excess liquid was blotted by attached blotting papers for 2 s to produce a thin sample film that was immediately vitrified by plunging to liquid ethane. The grid with the sample cryogenically immobilized was transferred onto a single tilt cryo-specimen holder for imaging.