Light scattering and passive mitochondrial swelling assay

Mitochondria from 10 male flies per group were isolated and processed for light scattering and swelling according to the procedure used by Navarro et al. (2004) [20]. Flies were homogenised using a pre-chilled mitochondrial storage buffer containing 230mM Mannitol, 70mM sucrose, 1mM EGTA, 10mM HEPES and protease inhibitor cocktail. The homogenate was then centrifuged at 800 x g for 30 min at 4°C and then the supernatant at 11,000 x g for 10 mins to precipitate the mitochondria. Mitochondrial suspensions, containing about 100ug protein/ml was subjected to swelling using a hypotonic solution (90mM KCl, 20mM MOPS-KOH, pH 7.2) and the absorbance changes were measured spectrophotometrically at 540nm for 5 min. The absorption rate (ΔA) and the initial rate (1 min) of mitochondrial swelling (ΔA/min) were recorded. The protein concentration of each sample was measured using the BCA assay (ThermoFisher Scientific).