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

The mitochondrial membrane potential (Delta Psi(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 Delta Psi(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 DiOC6(3) in the fluorometer was shown to be feasible for monitoring variations in Delta Psi(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 mu 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 DiOC6(3) in the Agilent 2100 bioanalyzer system for yielding sufficient fluorescence intensity in the mitochondria of the cells was 20 nm-2.0 mu m. Furthermore, significant reduction of fluorescence intensity in the cells stained with 2.0 mu m DiOC(6)(3) was observed after treatment with 10 mu m FCCP for 30 min. These results indicate that the Agilent 2100 bioanalyzer is potentially useful for monitoring Delta Psi(m) in cell assays.