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  • Title: A high-throughput assay for mitochondrial membrane potential in permeabilized yeast cells.
    Author: Farrelly E, Amaral MC, Marshall L, Huang SG.
    Journal: Anal Biochem; 2001 Jun 15; 293(2):269-76. PubMed ID: 11399043.
    Abstract:
    A fluorometric assay for mitochondrial membrane potential in permeabilized yeast cells has been developed. This method involves permeabilizing the plasma membrane and measuring the distribution of a mitochondrial membrane potential sensitive probe 3,3'-dipropylthiadicarbocyanine iodide (DiSC(3)(5); DiSC(3)). In permeabilized cells, DiSC(3) fluorescence decreased when introduced into energized mitochondria and increased three- to sixfold when the mitochondrial membrane potential was dissipated by the chemical uncoupler carbonylcyanide m-chlorophenyl hydrazone. Plasma membrane potential was abolished by permeabilization, as shown by a lack of polarization of the plasma membrane induced by K(+) and glucose. Uncoupling protein 1 (UCP1), a mitochondrial H(+) transporter, was used as a model for method validation. The fluorescence intensity responded vigorously to specific modulators in UCP1-expressing cells. This method has been adapted as a high-throughput assay to screen for modulators of mitochondrial membrane potential.
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