Measuring Mitochondrial Respiration in Frozen Liver Samples

The oxygen consumption rates (OCRs) in homogenates from frozen livers were measured with a Seahorse Extracellular Flux (XF) ‐96 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Frozen liver tissues were thawed and washed two times with ice-cold PBS. The ‘Respirometry in Frozen Samples (RIFS)’ protocol was adapted from Acin-Perez R et al. 2020.^{44 (link)} Approximately, 50 mg of tissue was cut into small pieces and homogenized in MAS buffer (70 mM sucrose, 220 mM mannitol, 5 mM KH₂PO₄, 5 mM MgCl₂, 1 mM EGTA, 2 mM HEPES, pH 7.4) with 20 strokes in a glass Dounce homogenizer. The homogenate was centrifuged at 1,000 g for 10 min at 4°C, the supernatant was collected and protein concentration was determined by Pierce^TM BCA assay (Thermo Fisher Scientific, Rockford, IL, USA). In Seahorse XF96 microplate, 10 µg (proteins) of homogenate was loaded into in 20 µl of MAS. The loaded plate was centrifuged at 2,000 g for 5 min at 4°C and an additional 130 µl of MAS containing cytochrome c (10 µg/ml) was added to each well. Substrates were delivered by port A (pyruvate + malate (5 mM each)) or NADH (1 mM), or 5 mM succinate + rotenone (5 mM + 2 µM). The inhibitors, Rotenone and antimycin A (2 µM + 4 µM) were delivered by port B. TMPD + ascorbic acid (0.5 mM + 1 mM) at port C; and azide (50 mM) at port D.