DYm Mitochondrial Membrane Potential
Example from Paul K.Y. Wong, Ph.D.& Na Liu, M.D., Research Core 1: Mechanisms of Toxicity and Cell Death
In the above example the Jurkat cell line is either untreated or treated with 2uM camptothecin (8 hours) and labeled with the dye JC-1 (Molecular Probes cat. # T-3168: ) to measure depolarization of the mitochondrial transmembrane electrical potential. The green fluorescent emission of single JC-1 molecules was collected through a 525nmBP filter with a 600nm dichroic to collect the orange fluorescent emission of JC-1 aggregates with a 610nmBP filter.
Oxidative phosphorylation across the mitochondrial membrane due to respiratory chain complexes generates electrons forming a transmembrane electrochemical potential or gradient of approx. 180mV - 200mV. This energy source also pumps protons (H+) that drive ATP synthesis. Researchers are interested in changes in mitochondrial membrane potential (DYm) and the role it plays in apoptosis. One theory is that a collapse in the mitochondrial transmembrane electrical potential is an early event and possible cause of programmed cell death. The lipophilic cation JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) binds to the polarized mitochondrial membrane as aggregates (from as low as ~ 80mV). The JC-1 aggregates upon excitation with an argon laser ( 488nmEX) have an emitted fluorescence at ~ 590nmEM (orange). Upon depolarization of the mitochondrial membrane the JC-1 forms monomers that emit fluorescence at ~ 530nmEM (green). Therefore there is a reversible shift from orange to green upon mitochondrial membrane depolarization or decrease in the transmembrane electrical gradient potential as JC-1 aggregates form monomeric JC-1. And likewise there is a reversible shift from green to orange upon increase in polarization or DYm.
References
Bradbury, D.A., Simmons, T.D., Slater, K.J., and Crouch, S.P.M. (2000) Measurement of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis. J. Immunol. Meth. 240:1-2 p. 79-92
Robinson, J. Paul, Mng. Ed.: Current Protocols in Cytometry 1998, John Wiley & Sons, Inc. New York, NY
Salvioli, S., Ardizzoni, A., Franceschi, C., and Cossarizza, A., (1997) JC-1, but not DiOC6(3) or Rhodamine123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 411: p. 77-82
Petit, P.X., Lecoeur, H., Zorn, E., Dauguet, C., Mignotte, B., and Gougeon, M. (1995) Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J. Cell Biol. 130:1 p. 157-167
Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, A., Hirsch, T., Susin, S.A., Petit, P.X., Mignotte, B., and Kroemer, G. (1995) Sequential Reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J. Exp. Med. 182:2 p. 367-377
Reers, M., Smiley, S.T., Mottola-Hartshorn, C., Chen, A., Lin, M., and Chen, L.B. (1995) Mitochondrial membrane potential monitored by JC-1 dye. Meth. Enzymol. 260: p. 406-417
Cossarizza, A., Baccarani-Contri, M., Kalashnikova, G., and Franceschi, C. (1993) A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Biochem. Biophys. Res. Commun. 197: p. 40-45
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