Analysis of mitochondrial membrane potential in the cells by microchip flow cytometry.
The mitochondrial membrane potential (DeltaPsi(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 DeltaPsi(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 DeltaPsi(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 microM 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 microM. Furthermore, significant reduction of fluorescence intensity in the cells stained with 2.0 microM DiOC(6)(3) was observed after treatment with 10 microM FCCP for 30 min. These results indicate that the Agilent 2100 bioanalyzer is potentially useful for monitoring DeltaPsi(m) in cell assays.
Kataoka
Masatoshi
M
Institute for Genome Research, The University of Tokushima, Japan. kataoka@genome.tokushima-u.ac.jp
Fukura
Yoko
Y
Shinohara
Yasuo
Y
Baba
Yoshinobu
Y
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002232:Carbocyanines; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D019169:Jurkat Cells; D008297:Male; D008564:Membrane Potentials; D046210:Microfluidic Analytical Techniques; D008930:Mitochondria, Liver / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D012402:Rotenone / Q000494:pharmacology; D019802:Succinic Acid / Q000494:pharmacology
D002232:Carbocyanines; D005456:Fluorescent Dyes; D012402:Rotenone; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C007392:3,3'-dihexyl-2,2'-oxacarbocyanine; D000244:Adenosine Diphosphate; D019802:Succinic Acid