Extraction of three-fraction components from hippocampal tissue homogenate by density gradient centrifugation

The synaptosome preparation protocol was adapted from a previously published method. Briefly, mice were euthanized by decapitation, and hippocampal tissues were homogenized in 500 μL of cold sucrose/EDTA buffer (0.32 M sucrose, 1 mM EDTA, 5 mM Tris, pH 7.4) containing 5 μL of a 100× protease/phosphatase inhibitor cocktail (Solarbio). Homogenization was performed using an electric homogenizer. The homogenate was centrifuged at 1,000 × g for 10 minutes at 4°C. The pellet (P1), containing cell debris and nuclei, was collected, while the supernatant (S1) was separated. Both P1 and 100 μL of S1 were stored at −80°C. The remaining S1 was layered onto a 10% Percoll density gradient (10% and 23% Percoll; supplemented with 0.32 M sucrose, 1 mM EDTA, 0.25 mM DTT, and 5 mM Tris, pH 7.4) and centrifuged at 31,000 × g for 10 minutes at 4°C in a SW-55Ti rotor (Beckman). The synaptosome fraction, located between the 10% and 23% Percoll layers, was carefully extracted using a pipette and transferred to a 1.5 mL centrifuge tube. The synaptosomes were washed with 1 mL PBS and centrifuged at 12,000 rpm. The resulting pellet represented the crude synaptosome fraction.