Simultaneous detection of mitochondrial respiratory chain activity and reactive oxygen in digitonin-permeabilized cells using flow cytometry.
BACKGROUND
Increased mitochondrial generation of reactive oxygen intermediates (ROI) due to defective respiratory chain activity has been implicated in physiological processes such as apoptosis, in the pathogenesis of mitochondrial diseases, and as part of the normal aging process. Established methods addressing activity of the respiratory chain complexes have been limited to bulk assays for single parameters. This study describes a flow cytometry-based method and its validation for the detection of respiratory chain function in single cells permeabilized by digitonin.


METHODS
Flow cytometry was used to measure mitochondrial membrane potential (DeltaPsi(m)) and reactive oxygen generation under differing conditions of respiration. This was brought about by the addition of substrates and inhibitors to digitonin-permeabilized cells. This method was validated by measurement of oxygen consumption and ATP production and by confocal microscopy.


RESULTS
Activity of the respiratory chain complexes assessed by DeltaPsi(m) responded to substrates and inhibitors as predicted from assessment by oxygen consumption and ATP synthesis. In addition, the flow cytometry method allows the simultaneous assessment of mitochondrial ROI generation. This was confirmed by the localization of the ROI probe, carboxy-DCF, to the same 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 the relationship between disordered function of respiratory chain complexes and mitochondrial ROI generation.
Pham
N A
NA
Division of Experimental Therapeutics, Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Canada.
Robinson
B H
BH
Hedley
D W
DW
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D017209:Apoptosis; D002463:Cell Membrane Permeability / Q000187:drug effects*; D019069:Cell Respiration; D004072:Digitonin / Q000494:pharmacology*; D004579:Electron Transport; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015203:Reproducibility of Results; D019802:Succinic Acid / Q000378:metabolism; D014407:Tumor Cells, Cultured
D005452:Fluoresceins; D008293:Malates; D017382:Reactive Oxygen Species; C030298:malic acid; D000255:Adenosine Triphosphate; D019802:Succinic Acid; D004072:Digitonin