Synaptosomes were extracted from iPSC-derived iNs at day 40 using Syn-PER synaptic protein extraction reagent (Thermo Fisher Scientific, catalog #87793) and conjugated with pHrodo red amine-reactive labels (Life Technologies, catalog #P36600) following the manufacturer’s instructions. Syn-PER reagent (400 μl for one well in six-well plates) was added to iNs. Cells were scraped to collect lysate and then centrifuged at 1200g for 10 min at 4°C. The supernatant was transferred to a new tube and centrifuged at 15,000g for 20 min at 4°C to retain the synaptosome pellet. The synaptosome pellet (200 μg) was suspended in 100 μl of 0.1 M NaHCO3 (pH 8.3) and 1 μl of pHrodo red dye. The suspension was then incubated on a twist shaker at 40 rpm for 1.5 hours at room temperature, covered with aluminum foil. After incubation, ice-cold Dulbecco’s PBS (DPBS) was added, followed by centrifugation at 13,000 rpm for 2 min at 4°C. The pellet was washed five times with DPBS to remove unbound dye. Last, the pellet was resuspended in 100 μl of DPBS with 5% dimethyl sulfoxide.
