Isolation and Characterization of Synaptosomes

Synaptosomes were isolated using Syn-PER Reagent (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s protocol. Briefly, approximately 30 mg of tissue was homogenized using Dounce glass homogenizer in the presence of Halt Protease Inhibitor Cocktail (ThermoFisher Scientific, Waltham, MA) and Phosphatase Inhibitor Cocktail (MilliporeSigma, Burlington, MA). The homogenate was spun down at 1200×g for 10 min at 4 °C. The supernatant was centrifuged at 15,000×g for 20 min at 4 °C to obtain the pellet of synaptosomes. The pellet was then resuspended in HBK (HEPES-buffered Krebs-like) buffer as described before [24 (link)]. The concentration of synaptosomes was determined using flow cytometry. The samples were stored at − 80 °C until use. Synaptosome preparations are routinely analyzed by Western blot and electron microscopy to ensure the quality of the preparation, as we have previously reported [24 (link)].