2.14. Mitochondrial ATP Production Determination

ATP production, a primary function of mitochondria, was determined using an ATP determination kit (Thermo Fisher Scientific, Waltham, MA, USA) following a modified protocol. To measure the real-time production of ATP from isolated mitochondria, 1 mM malate, 2 mM ADP, and 1 mM pyruvate were incubated with 5 μg of mitochondria. The ATP produced was measured using the kit components according to the manufacturer’s protocol. Luminescence intensity, coupled with ATP production, was monitored using SpectraMax iD3 (Molecular Devices, San Jose, CA, USA).