Analysis of mitochondrial membrane potential in the cells by microchip flow cytometry

The mitochondrial membrane potential (ΔΨ m ) is an important indicator of the energetic state of both the mitochondria and the cells. To develop a sensitive, convenient, and rapid method for the measurement of ΔΨ m , we carried out cell fluorescence assays using the Agilent 2100 bioanalyzer system which, unlike the conventional flow cytometry, is based on microfluidic technology employing fluorescence detection with a 3,3'‐dihexyloxacarbocyanine iodide (DiOC 6 (3)) fluorescent probe. The use of DiOC 6 (3) in the fluorometer was shown to be feasible for monitoring variations in ΔΨ m in the mitochondria isolated from rat liver and treated with rotenone, succinate, ADP, and carbonylcyanide‐ p ‐trifluoromethoxyphenylhydrazone (FCCP). Flow cytometry analysis showed severe reduction of fluorescence intensity in Jurkat cells after treatment with 1.0 and 10 μ M FCCP. However, fluorescence microscopy demonstrated obvious accumulation of fluorescence in the mitochondria and induction of diffuse cytoplasmic fluorescence not localized to the mitochondria in these cells. The dose response range of DiOC 6 (3) in the Agilent 2100 bioanalyzer system for yielding sufficient fluorescence intensity in the mitochondria of the cells was 20 nm‐2.0 μ M . Furthermore, significant reduction of fluorescence intensity in the cells stained with 2.0 μ M DiOC 6 (3) was observed after treatment with 10 μ M FCCP for 30 min. These results indicate that the Agilent 2100 bioanalyzer is potentially useful for monitoring ΔΨ m in cell assays.