2.1.6. Mitochondrial permeability transition pore (MPTP) opening assay

MPTP opening in SSM and IFM was performed as previously described [20], [24]. Brief, mitochondria (500 µg) were resuspended in 2.0 mL assay medium [containing 100 mM KCl, 50 mM MOPS, 5 mM KH₂PO₄, 5 mM EGTA, 1 mM MgCl₂, 5 mM glutamate, and 5 mM malate] and assayed for tert-butyl-induced pore opening in a fluorescence spectrophotometer at 37 °C. Calcium uptake was taken from the fall in extramitochondrial Ca²⁺ following a bolus injection of 3 μL of 15 mM Ca²⁺ (30 nmoles Ca²⁺/mg mitochondrial protein) and tert-butyl-induced calcium release were assessed in both isolated subpopulations SSM and IFM after calcium uptake. Tert-butyl hydrogen peroxide (400 mM) was infused at a rate of 0.2 μL/min, and the concentration of free Ca²⁺ in the medium was calculated by monitoring the fluorescence of the Ca²⁺ indicator calcium green-5N (CaGN-5N; Molecular Probes) with an excitation and emission of 488 and 530, respectively. MPTP opening was inferred from the sudden and large increase of fluorescence.