The CRC assay and the mitochondrial swelling assay were performed simultaneously using a dual-detector (one to measure fluorescence and the other to measure absorbance), single-cuvette–based fluorimetric system (Horiba Scientific). Depending on the experiment, 1 or 2 mg of isolated mitochondria was loaded into the cuvette along with 250 nM Calcium Green-5N (Invitrogen), 7 mM pyruvate (Sigma-Aldrich), and 1 mM malate (Sigma-Aldrich) and brought up to 1 ml using KCl buffer. Mitochondria were then pulsed with sequential additions of CaCl2 (40 or 800 μM) until MPTP opening occurred or until the mitochondria reached a CaCl2 saturation point and could no longer take up more Ca2+. In certain situations where the experimental mitochondria did not undergo a swelling event following CaCl2 additions, they would be subjected to a membrane-permeabilizing agent, 40 μM alamethicin (Santa Cruz Biotechnology), to show that they still had swelling capacity and were not otherwise compromised.

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