Procedure

1. Add 1 mg/ml freshly isolated mitochondria in assay buffer to a 96-well

U-bottom plate with or without S100, in a final volume of 60 ^l.

Pretreat with any PT inhibitors (i.e. 10 ^M cyclosporin A, 30 min) as necessary before incubating the mitochondria with the PT inducers at 370C for the desired time period, usually within 0-4 h.

3. Centrifuge the plate at 760g, 5 min. Add 30 ^l of supernatant to 7.5 ^l of 5x SDS loading buffer. Boil for 5 min then add 10 ^l to a standard 15% SDS-PAGE gel for Western blotting. Use antibodies recognizing cytochrome c to verify cytochrome c release into the supernatant and mitochondrial dysfunction. C1

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