Luciferase atp assay kit

Renal tissue adenosine triphosphate (ATP)was measured by Luciferase ATP Assay Kit (Beyotime, China) according to the manufacturer’s instructions. Briefly, 20 mg tissues were lysed with 200 μl lysis buffer on ice bath, and then centrifuged 12000g for 5 minutes at 4 °C. 100 μl of supernatant was collected and mixed with 100 μl ATP detection solution. Luminance was measured by fluorescence microplate reader (Synergy4, BioTek, USA). The ATP level was normalized to the protein concentration of each sample.

ATP levels were measured using a luciferase ATP assay kit (Beyotime, China). Briefly, 200 μL of lysis buffer was added to cells treated or untreated with Al₂O₃ NPs. Cells were collected and centrifuged at 12 000 rpm for 5 min at 4 °C. 0.05 g lung tissue were homogenized with 250 μl lysis buffer, then centrifuged at 12,000 rpm for 5 min at 4 °C. The luminescence of the supernatant was assayed by a luminometer (Berthold Detection System, Pforzheim, Germany). The cationic dye JC-1 was used to detect the mitochondrial membrane potential, and HBE cells exposed to Al₂O₃ NPs 12 or 24 h were evaluated under a fluorescence microscope (Olympus, Japan) to examine green and red fluorescence.
To determine the release of cytochrome c, mitochondrial and cytosolic proteins were isolated by a Mitochondria/Cytosol Fractionation Kit (Beyotime, China) according to the manufacturer’s instructions. The protein concentrations in cytosol and mitochondria samples were measured using the Bradford method. The levels of cytochrome c were estimated according to the ELISA kit procedures (R&D Systems, U.S.). For all of these mitochondrial-involved assays, CCCP was employed as positive control.