Seahorse Measurement of Mitochondrial Oxygen Consumption Rates

Isolated mitochondria (1 μg/100 μL) from the hearts of Sham and post-CPR mice were suspended in 24-well plates. Oxygen consumption rates (OCRs) were determined using the Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA), as previously described (21). Complex I OCR was measured using the substrates 10-mM pyruvate + 2-mM malate. Complex II OCR was measured using the substrate 10-mM succinate and an inhibitor of reverse electron flow, 2-µM rotenone. Additional details are described in the supplemental methods (Supplemental Digital Content 1, http://links.lww.com/CCM/F61).