Benchtop mitochondria isolation kit

Neonatal mouse cardiomyocytes mitochondria were isolated using Abcam’s benchtop mitochondria isolation kit. After frozen and thawed to weaken membranes, cells were suspended in Reagent A at 5 mg/mL on ice for 10 min. Then cells were homogenized using a glass Dounce homogenizer, and centrifuged at 1000 × g for 10 min at 4°C twice. The supernatant was then centrifuged at 12000 × g for 10 min at 4°C. The remaining pellet was resuspended in Reagent C with phosphatase/protease inhibitor and frozen at −80°C. Mitochondrial protein concentration was detected using Enhanced BCA Protein Assay Kit (Beyotime Biotechnology).

Mitochondria were isolated from NMCMs using Abcam's benchtop mitochondria isolation kit. Cells were frozen and thawed to weaken membranes, suspended in Reagent A at 5.0 mg/ml, incubated on ice for 10 min. Then NMCMs were homogenized with a glass Dounce homogenizer, and were then centrifuged at 1000× g for 10 min at 4 °C twice. The supernatant was centrifuged at 12000× g for 10 min at 4 °C, and the remaining pellet was resuspended in Reagent C supplemented with phosphatase/protease inhibitor and frozen at -80 °C. Mitochondrial protein concentration was determined by colorimetry using Enhanced BCA Protein Assay Kit (BCA).