Luminescence plate reader

Activity of Cytochrome c oxidase, a key enzyme of respiratory chain complex IV, and ATP levels were determined as described previously [17 (link)]. Briefly, cybrid cells were washed with ice-cold PBS, and then harvested, centrifuged, and suspended in 50 μl of isolation buffer containing 250 mM sucrose, 20 mM HEPES, and 1 mM EDTA. Cell suspensions (containing 3–4 mg of protein/ml) were added to a cuvette containing 0.95 ml of 1 × assay buffer (10 mM Tris-HCl, pH 7.0 and 120 mM KCl), and the reaction volume was brought to 1.05 ml with the addition of 1 × enzyme dilution buffer (10 mM Tris-HCl, pH 7.0 and 250 mM sucrose). The reaction was then initiated by the addition of 50 μl of ferrocytochrome substrate solution (0.22 mM). The rate of change in absorbance at 550 nm was recorded immediately using a Shimadzu (Kyoto, Japan) UV1200 spectrophotometer programed for a 5 s delay and 10 s intervals for 6 readings. ATP levels were determined using an ATP Bioluminescence Assay Kit (Roche) following the manufacturer’s instruction as we previously described [8 (link)]. Briefly, cells were quickly harvested by ATP lysis buffer, incubated on ice for 30 min, and then centrifuged at 12,000 g for 10 min. ATP levels were measured by Luminescence plate reader (Molecular Devices). A 1.6 s delay time after substrate injection and 10 s integration time were used.

ATP levels in cortex were determined using an ATP Bioluminescence Assay Kit (Roche) following the manufacturer’s instruction. Mice brain tissues were homogenized in lysis buffer provided in the kit, incubated on ice for 15 min, and centrifuged at 14,000 g for 15 min. Subsequent supernatants were measured for the ATP levels using a Luminescence plate reader (Molecular Devices) with an integration time of 10 s.