Quantifying Mitochondrial Permeability Transition

Opened mPTPs of the quadriceps muscle were measured by fluorexon with the mPTP assay kit (Genmed Scientifics Inc., Wilmington, DE, USA), in accordance with the manufacturer's instructions. Briefly, 0.1 mL of depurated mitochondrial suspension, containing 200 μg protein, was added into Reagent A; this mixture was incubated at 37°C for 15 min and centrifuged at 16000 × g for 5 min. The supernatant was discarded and the precipitate was resuspended with Reagent C; the fluorescence intensity of the reaction medium was determined using Varioskan Flash (Thermo Fisher Scientific), with excitation wavelength (λ_ex) 488 nm and emission wavelength (λ_em) 505 nm.