Isolated mitochondria or mitoplasts (100 μg of proteins) from 4-month-old mice were fixed in 2% formaldehyde for 15 min, centrifuged at 10,000g for 10 min and rinsed with PBS. The pellet was then resuspended and incubated 10 min at room temperature in 100 μl of PBS supplemented with 35 μM of NAO. Mitochondria or mitoplasts were then centrifuged at 10,000g for 10 min, and pellets were resuspended in 100 μl of PBS and fluorescence (λ excitation, 495 nm; λ emission, 620 nm) was acquired in 96-well plates with a spectrofluorimeter (TECAN infinite M200, TECAN, Austria).

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