Luminescence enhanced atp assay kit

The concentration of ATP in isolated mitochondria was detected using a luciferase-based luminescence enhanced ATP assay kit (Beyotime). Isolated mitochondria (1 mg/mL protein) were incubated in respiration buffer (0.5 mL) with 2.5 mM malic acid, 2.5 mM succinate, and 2.5 mM ADP for 10 min. The ATP concentrations in the mitochondrial suspension and cell lysates were determined using a SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices, Sacramento, CA, USA).

We used the luciferase-based luminescence enhanced ATP assay kit (Beyotime, Shanghai, China) to measure the level of adenosine triphosphate (ATP) in the isolated mitochondria. We incubated isolated mitochondria (1 mg/ml) into the 0.5-mL respiration buffer (2.5 mM succinate, 2.5 mM malic acid, and 2.5 mM ADP) for 10 min. We used the SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices, Sacramento, CA, USA) to detect the ATP concentrations.

The H9c2 cell line was purchased from the cell bank of the Institute of Biochemistry and Cell Biology of Shanghai (Shanghai, China) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin streptomycin (Gibco, Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazoliumbromide (MTT) was bought from Sigma Aldrich Inc. (St Louis, MO, USA). Doxorubicin and luminescence-enhanced ATP assay kit were purchased from Beyotime Biotech Inc. (Shanghai, China). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) was obtained from Thermo Fisher Scientific Inc. (Rockford, IL, USA). DNA extraction kit (TIANGEN, Beijing, China), Long Amp Taq 2× Master Mix (NEB, Ipswitch, MA, USA) and Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) were used for mitochondrial DNA damage assay. Primary antibodies for p-JNK, JNK, Mfn1, and Mfn2 were purchased from Abcam Inc. (Cambridge, MA, USA). Primary antibodies for β-actin and caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA), HRP goat anti-rabbit antibody and HRP goat anti-mouse antibody were purchased from Beijing BioDee Biotechnology Co., Ltd. (Beijing, China).