Mitocheck complex activity assay kit

The mitochondrial functions of U87 cells at a density of 5.0×10⁴ viable cells/cm² cultured with 0, 1, 10 and 20 µM Aβ_1–42 in a cell culture incubator at 37°C for 48 h was detected. ATP generation was detected using an ATP assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Briefly, 200 µl lysis buffer was added to U87 cells cultured in 6-well plates, which were subsequently centrifuged at 12,000 × g at 4°C for 5 min. The supernatant was collected and detected together with ATP standard solution on the luminometer. Mitochondrial membrane potential (MMP) was detected using a JC-1 staining kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. In addition, the activity of the four complexes of the electron transport chain (ETC) following treatment of U87 cells with 10 µM Aβ_1–42 for 48 h were also detected using MitoCheck Complex Activity Assay Kit (I: 700930, II: 700940, III: 700950 and IV: 700990; Cayman Chemical Company, Ann Arbor, MI, USA, Catalog No.). The rate of NADH oxidation was measured at 340 nm for the activity of complex I. A decrease in absorbance at 600 nm for complex II activity, the reduction of excess cytochrome c (550 nm absorbance) for complex III and the direct oxidation of cytochrome c for complex IV. Results were presented relative to values in the control (0 µM) group.