Mitochondrial ATP and ATP synthase activity measurement

Mitochondrial ATP was quantitated by CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. In brief, mitochondria were isolated from cells and resuspended in mitochondrial buffer that contained 2% of trichloroacetic acid. Equal amounts of mitochondria were subjected to luminescence reading in the SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA, USA). Mitochondrial ATP synthase activity was measured by using the ATP Synthase Enzyme Activity Microplate Assay Kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s protocol. Equal amounts of mitochondria were loaded onto the provided plate and incubated at room temperature. Wells were washed twice and incubated at room temperature with the provided lipid mix. Reagent mix was added and the plate was read at OD 340 nm and expressed as ΔOD₃₄₀/min.