Luciferin luciferase bioluminescence assay kit

The activity of the mitochondrial complex I and III were estimated by measuring the produced amount of ROS and ATP production in hippocampal mitochondrial enriched preparations. Mitochondrial ROS production was measured using 25 μM of CM-H2DCFDA (DCF) (485 nm, 530 nm) in the Biotek Synergy HT plate reader as previously described^{31 (link),43 (link),44 (link)}. Isolated mitochondria (25 μg of protein) were added to 100 μl of KCl respiration buffer with 5 mM pyruvate and 2.5 mM malate as oxidative substrates and incubated at 37 °C. ROS production was calculated as the maximum DCF fluorescence following 30 min of incubation, expressed in arbitrary fluorescence units. After ROS measurements, KCl respiration buffer containing mitochondria was centrifuged at 8000 g for 10 min at 4 °C and then the ATP concentration was measured in the supernatant using the luciferin/luciferase bioluminescence assay kit (ATP determination kit no. A22066, Molecular Probes, Invitrogen)^{31 (link),43 (link)–45 (link)}.

ATP was measured using a luciferin/luciferase bioluminescence assay kit (Molecular Probes). The intensity of chemiluminescent signal was determined by a luminometer (Gene Light 55; Microtech Nition, Chiba, Japan) as described previously [7 (link),22 (link),23 (link),29 (link),30 (link),31 (link)].