Sterile pbs ph 7

Plasma prep or CCM was centrifuged at 12,000 × g for 20 min to remove cellular debris. The supernatant was then ultracentrifuged at 100,000 × g for 90 min to pellet the EVs using 36 mL Polyethylene terephthalate (PET) tubes (Thermo Fisher Scientific). Excess tube volume was filled with sterile PBS pH 7.4 (Gibco). The extracellular vesicle-pellet was washed with PBS and centrifuged at 100,000 × g for 90 min. Extracellular vesicles were suspended in 100 μL PBS pH 7.4 or RIPA with protease inhibitor cocktail (Thermo Fisher Scientific) and frozen at −20°C. EVs stored in PBS were then used for nanoparticle tracking analysis along with RNA extraction and characterization, while EVs in RIPA were used for western blot analysis.

After α-Syn sedimentation, PFF were diluted to 2 mg/ml in sterile PBS pH 7.4 (Gibco) and subjected to ultrasound breakage for 20 min using a Sonifier (Branson 250; 30% intensity) equipped with a water jacket cooling system to avoid sample overheating. Then, the size distribution profile of PFF in suspension was measured by DLS using a Wyatt DynaPro NanoStar instrument at 25 °C. Data were processed using the Dynamics 7.5.0.17 software package with the solvent (PBS) background signal subtracted from each sample.