Calcium retention capacities of rat CH mitochondria were measured as previously described (16) with some modifications. Briefly, primary CHs were incubated with 0.25 M sucrose buffer for complex 1 without EGTA at 25°C (room temperature). Extra-mitochondrial calcium was visualized using a 500 nm excitation wavelength and emission wavelengths of 530 nm with 1 μM calcium green -5N. Experiments were performed in the presence and absence of 1.2 mM MgCl2 and 40 μM ADP, as well as 1 mM cyclosporine A (CsA), an inhibitor of the mitochondrial permeability transition pore (MPTP). The probe reversibly binds to calcium ions. The inclusion of CsA allows for an increased Ca2+ retention capacity prior to induction of the mPTP and the subsequent release of Ca2+ from the mitochondria.

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