ADP/ATP Exchange Assay for Mitochondria

Mitochondria were isolated according to a previous report^{13 (link)} with some modifications (see Suppl. Materials and Methods). The ADP/ATP exchange assay was carried out in a filter plate format using a 96-well MultiScreenHTS FC filter plate (Millipore, Billerica, MA). Compound or DMSO diluted into mitochondria isolation buffer (MIB) (20 mM HEPES-KOH, 0.6 M mannitol, pH 7.4) was transferred into the precooled plate (50 μL/well). Mitochondria were added in 25 μL MIB (50 μg/well) and incubated for 10 min. ADP/ ATP exchange was initiated by adding 25 μL of 20 μM [³H] ADP (5 μCi/mL). The exchange reaction was then stopped after 1 min by the addition of 25 μL cATR (5 μM final). The reaction mix in the filter plate was then through on a vacuum manifold, and the plate was washed four times with 300 μL/well MIB buffer. Supermix scintillation cocktail (PerkinElmer, Waltham, MA) (100 μL) was transferred into each well and incubated overnight, and the [³H]ADP taken up by mitochondria was measured with a MicroBeta scintillation counter (PerkinElmer).