Determination of the mitochondrial membrane potential ψm

The membrane potential of the inner mitochondrial membrane was measured using Rhodamine 123 dye following previous report (68). The mice were sacrificed by decapitation, and the brain cerebral cortex was quickly removed and minced in the suspension buffer (138 mM NaCl, 5.4 mM KCl, 0.17 mM Na₂HPO₄, 0.22 mM K₂PO₄, 5.5 mM glucose, and 58.4 mM sucrose, pH 7.35), and the cells dissociation was further achieved by trituration through 18 and 21 gauge needles. The resulted suspension was then filtered through 100 μm cell strainer (BD Bioscience, San Jose, CA), and the cells in filtrate were collected after 5min centrifugation at 400 g at 4 °C after washing with wash buffer (110 mM NaCl, 5.3 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 25 mM glucose, 70 mM sucrose, 20 mM HEPES, pH 7.4). The cells were then suspended in Dulbecco’s modified Eagle’s medium, which was pre-adapted in 5% CO₂ tissue culture incubator for 30min. For the mitochondrial membrane potential assay, 250 mu;l of cell suspension (1×10⁷ cells/ml) was incubated with 0.4 μM Rhodamine 123 in 48-well plate for 15min at 37 °C in 5% CO₂ incubator. The fluorescence was recorded at λex490nm/em535nm using a Tecan plate reader (Tecan, Switzerland) after washing three times with Hank’s balanced salt solution (ThermoFisher, Grand Island, NY).