Measurement of mitochondrial respiration

Seahorse XF24 Extracellular Flux Analyzer (Seahorse Biosciences, North Billerica, MA) was used to assess the mitochondrial respiratory function. Freshly isolated mitochondria (10 μg/well) from control and treated mouse pancreas were plated onto poly-l-lysine coated Seahorse plates. Mitochondria were energized by adding 8 mM succinate. Respiration was sequentially measured in energized mitochondria (basal respiration), followed by state 3 (phosphorylating respiration), in the presence of 4 µM ADP, state 4 (resting respiration) after addition of 2.5 μg/ml oligomycin, an inhibitor of mitochondrial ATPase. Uncoupled maximal respiration was determined by adding 150 μM DNP or 4 μM FCCP as mentioned in figure legends. Finally, complex III inhibitor antimycin A (4 μM) was added for measuring non-mitochondrial respiration. Results represent O₂ consumption/min/µg mitochondrial protein.