Measuring Mitochondrial Respiration in Mouse Hearts

Mitochondria were isolated from freshly excised hearts of 2-month-old-mice and oxygen consumption rate was measured using a Seahorse Extracellular Flux Analyzer (Agilent Technologies, XF24) as described previously (Huo et al., 2020 (link)). Briefly, 25 μg of isolated heart mitochondria were loaded onto XF24 cell culture microplates (Agilent Technologies, #102342-100) in KCl buffer (125 mM KCl, 20 mM HEPES, 2 mM MgCl₂, 2 mM KH₂PO₄, and 40 µM EGTA, pH 7.2) containing 25 mM pyruvate, 10 mM malate and 5 mM ADP. Microplates were subjected to the XF Mito Stress Test Kit (Agilent Technologies, #103015-100) following the manufacturer’s standard protocol. After basal respiration was measured, the mitochondria were treated sequentially with 2 μM oligomycin, 5 μM carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 μM rotenone.