Phrodo ifl green

Synaptosomes were isolated from WT mice using Syn-PER Synaptic Protein Extraction Reagent (Thermo Scientific), per the manufacturer’s instructions. For pHrodo labeling, dissolved pHrodo iFL green (Life Technologies) was incubated with synaptosomes on a shaker in PBS for 1 h at room temperature protected from light at the ratio of 20 μg pHrodo per 1 mg synaptosomes. Unconjugated pHrodo was removed by washing with PBS before pHrodo-conjugated synaptosomes were resuspended in PBS with 5% dimethyl sulfoxide, aliquoted, and stored at −80 °C until use.

Primary mouse neurons were prepared from B6 embryos at E16.5 to E17.5. Cerebral hemispheres were isolated and freed from meninges before tissue digestion with 0.25% trypsin in HBSS for 15 min at 37 °C followed by titration to obtain single-cell preparations. Cell suspensions were filtered through a 70-μm cell strainer and cells were centrifuged at 600 × g for 5 min. Cell density was determined using a hemocytometer, and cells seeded in Neurobasal medium were supplemented with 1× B27 and 500 μM GlutaMAX (Invitrogen). Half of the medium was changed every 3 d. To induce apoptosis, primary cultured WT neurons were treated with 300 μM N-methyl-d-aspartate overnight before careful detachment from flasks by repeated washes with PBS followed by centrifugation, and the pellet was processed for labeling. Neurons (1 × 10⁷) were carefully resuspended in 1 mL PBS and incubated in darkness for 2 h at room temperature with 100 μg of dissolved labeling dye (pHrodo iFL green; Life Technologies). To block and capture residual dye, cells were diluted with PBS, harvested by centrifugation, resuspended in 1 mL undiluted fetal bovine serum (FBS), and washed twice with PBS. Total apoptotic cell numbers were determined using trypan blue staining.