Determination of mitochondrial integrity and functions

The cardiolipin-sensitive probe Nonyl Acridine Orange (NAO; Sigma-Aldrich) is able to monitor changes in mitochondrial lipids [64,70,71]. NAO, used at low concentrations in living cells, is an efficient fluorescent indicator for cardiolipin of inner mitochondrial membranes. In the present study, it was used to measure the mitochondria mass/number independently of mitochondrial membrane potential (ΔΨm). After an incubation of cells with 100nM NAO for 15 min at 37°C in the dark, samples were acquired by flow cytometry using the appropriate fluorescence channels. Mitochondria were also stained with MitoTracker Green (MTG) 100nM for 30 minutes at 37°C. Cells were then analysed by flow cytometry, collecting at least 10,000 events for each sample.

The ΔΨm was analysed using the (ΔΨm)-specific stain TMRE, a tetramethylrhodamine able to selectively enter into mitochondria according to the Nernst equation [72,73]. Cells were loaded with 40 nM TMRE at 37°C for 15 min and the analysis was carried out under nonquenching conditions. The specificity of this analysis was determined using the uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone; Sigma-Aldrich), which caused mitochondrial membrane depolarization with a sudden decrease of TMRE fluorescence. Cells were treated for 30 min with 10μM CCCP, stained with TMRE as described previously and then acquired by flow cytometry. Fluorescence intensity is expressed as the initial signal after background subtraction.