Synaptosome isolation and pHrodo labeling

Briefly, mice were anesthetized and forebrains were quickly removed and homogenized in ice-cold gradient buffer (320.0 mM sucrose, 5.0 mM HEPES, 0.1 mM EDTA, pH 7.5). The homogenate was centrifuged at 1000 × g for 20 min to collect supernatant, and supernatant was centrifuged at 1200 × g for 10 min to discard cell nuclei and debris and collect supernatant. The supernatant was centrifuged at 10,000 × g for 10 min to obtain pellet. The pellet was resuspended in gradient buffer and loaded onto a sucrose gradient (0.8 M: 1.2 M = 1: 1). The thin layer (the layer between 0.8 M sucrose and 1.2 M sucrose) was collected carefully after centrifugation at 100,000 × g for 1 h, and diluted with an equal volume of ultrapure water before centrifuged at 100,000 × g again to acquire the purified synaptosome pellet. The synaptosome pellet was resuspended in ice-cold artificial cerebrospinal fluid (aCSF, 2 mM CaCl₂, 132 mM NaCl, 3 mM KCl, 2 mM MgSO₄, 1.2 mM NaH₂PO₄, 10 mM HEPES, and 10 mM glucose, pH 7.4).

For pHrodo labeling, synaptosomes were incubated with pHrodo Red succinimidyl (NHS) ester (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36000","term_id":"549719","term_text":"P36000"}}P36000) or pHrodo Green STP ester (Life Technologies, {"type":"entrez-protein","attrs":{"text":"P36013","term_id":"547888","term_text":"P36013"}}P36013) in sodium carbonate buffer pH 9.0 for 2 h at 4 °C in the dark at the concentration of 1 μL pHrodo/1 mg synaptosomes. After incubation unconjugated pHrodo was washed by DPBS.