Flow-cytometric monitoring of mitochondrial depolarisation: from fluorescence intensities to millivolts.
Redistribution potentiometric dyes represent a powerful tool for monitoring membrane potential of mitochondria, especially when these dyes are used with flow cytometry. In particular, tetramethylrhodamine methyl ester proved to be suitable for the screening of mitochondrial membrane potential in cultured human skin fibroblasts from patients suffering from different defects of oxidative phosphorylation. We have developed a method that makes it possible to measure the changes in mitochondrial membrane potential, or to assess the differences between respective mitochondrial membrane potentials in investigated cells and controls in the absolute scale of millivolts. Our approach employs the fact that a logarithmic transformation of Nernst equation-controlled intensity of fluorescence from potentiometric dyes accumulated in mitochondria leads to a linear scale for mitochondrial membrane potentials.
Plásek
J
J
Faculty of Mathematics and Physics, Institute of Physics, Charles University, Ke Karlovu 5, 121 16 Prague, Czech Republic. plasek@karlov.mff.cuni.cz
Vojtísková
A
A
Houstek
J
J
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000447:Aldehydes / Q000494:pharmacology; D000818:Animals; D002138:Calibration; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D051381:Rats
D000447:Aldehydes; C111472:mitotracker green FM; D000244:Adenosine Diphosphate