Mitochondrial membrane potential provides a valuable indicator of cells' health and functional status. Cytometry- and microscopy-based analyses, in combination with fluorescent probes, are widely used to study mitochondrial behavior related to cellular pathways, most notably - apoptosis. The cyanine dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) facilitates discrimination of energized and deenergized mitochondria because the normally green fluorescent dye forms red fluorescent aggregates when concentrated in energized mitochondria in response to their higher membrane potential. JC-1 fluorescence is usually excited by the 488 nm laser wavelength common in flow cytometers. In this study, we show that in practice this approach is not optimal for monitoring mitochondrial behavior. Investigation of fluorescence of JC-1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard instruments, produces signals from aggregate fluorescence with considerably less spillover from dye monomer fluorescence than can be obtained using 488 nm excitation. The improved data are more accurate and eliminate the necessity for fluorescence compensation, making the use of the alternative excitation wavelengths beneficial for mitochondria-related biological and biomedial research.
|Journal||Cell Death and Disease|
|State||Published - 22 Nov 2012|
Bibliographical noteFunding Information:
Acknowledgements. We thank Dr. Joseph Orly for the valinomycin, and Dr. Roxane Lahmi, Dr. Manuela Vecsler, Dr. Ayala Sharp and Mr. Eitan Ariel for technical assistance. Funding by the Marie Curie International Reintegration Grant PIRG-GA-2010-277062 (AT), the Israeli Centers of Research Excellence (I-CORE), Gene Regulation in Complex Human Disease, Center No. 41/11 (AT), and the Israel Cancer Association Grant 20120067 (AT) is gratefully acknowledged.
- Cyanine dyes
- Flow cytometry
- Membrane potential