Simultaneous detection of mitochondrial respiratory chain activity and reactive oxygen in digitonin-permeabilized cells using flow cytometry

Background: Increased mitochondrial generation of reactive oxygen intermedi ates (ROT) due to defective respiratory chain activity has been implicated in physiological processes such as apoptosis, in the pathogenesis of mitoch ondrial diseases, and as part of the normal aging process. Established meth ods addressing activity of the respiratory chain complexes have been limite d to bulk assays for single parameters. This study describes a now cytometr y-based method and its validation for the detection of respiratory chain fu nction in single cells permeabilized by digitonin. Methods: Flow cytometry was used to measure mitochondrial membrane potentia l (Delta Psi (m)) and reactive oxygen generation under differing conditions of respiration. This was brought about by the addition of substrates and i nhibitors to digitonin-permeabilized cells. This method was validated by me asurement of oxygen consumption and ATP production and by confocal microsco py. Results: Activity of the respiratory chain complexes assessed by Delta Psi (m), responded to substrates and inhibitors as predicted from assessment by oxygen consumption and ATP synthesis. In addition, the now cytometry metho d allows the simultaneous assessment of mitochondrial ROI generation. This was confirmed by the localization of the ROI probe, carboxy-DCF, to the sam e site as the mitochondrial probe observed by confocal microscopy. Conclusions: This method allows the functional integrity of the respiratory chain complexes to be studied at the single-cell level, thus addressing th e relationship between disordered function of respiratory chain complexes a nd mitochondrial ROI generation. Cytometry 41.245-251, 2000. (C) 2000 Wiley -Liss, Inc.