Atp assay kit

The levels of ATP were measured using an ATP assay kit (Abnova, Taiwan) according to the manufacturer’s instructions. Briefly, mouse liver and ES cells were lysed in 100 μl of ATP buffer, and then centrifuged in ice-cold buffer at 15,000 g for 2 min. Supernatant was collected and 50 μl was added to a 96-well plate. Subsequently, reaction mix (50 μl) was added to each well before incubating at room temperature for 30 min in the dark. A standard curve was generated using different ATP concentrations (0, 2, 4, 6, 8, 10 nmol/well). In the ATP colorimetric assay, O.D. was measured at 570 nm. Total ATP levels were expressed as nmol/μl.

Intracellular ATP levels were measured by the ATP assay kit (AAT-15204, Abnova). 1 × 10⁴ hiPSCs were seeded in white opaque microplates. After 24 h, the culture medium was removed and changed into the Reconstituted Reagent (95 μL Assay Buffer with 1 μL Substrate and 1 μL ATP enzyme); 90 μL of Reconstituted Reagent was added to each well, and ATP production was measured on a Luminometer (Promage) within 1 min.

Protein was extracted using M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, Rockford, IL, USA). Human Cytochrome C ELISA Kit (Abcam, Cambridge, Cambridgeshire, UK) was used to quantify the mitochondrial activity. The production of lactate, ATP, and ROS was determined using the Lactate Assay Kit-WST (Dojindo, Kumamoto, Japan), ATP Assay Kit (Abnova), and Lipid Peroxidation (MDA) Assay Kit (Biovision), respectively.