Fluorescence membrane potential dyes

Fluorescence Assays Using Membrane Potential Dyes... 

Fluorescence-based methods do not directly measure ionic current but, rather, measure either membrane-potential-dependent or ion-concentration-dependent changes of fluorescence signals (from fluorescent dyes loaded into the cytosol or cell membrane) as a result of ionic flux. Because fluorescence-based methods give robust and homogeneous cell population measurement, these assays are relatively easy to set up and achieve high throughput. 

Mandala M, Serck-Hanssen G, Martino G, Helle KB (1999) The fluorescent cationic dye rhodamine 6G as a probe for membrane potential in bovine aortic endothelial cells. Anal Biochem 274 1-6... 

The method described here can be applied to gain insight about environmental effects on the absorption and fluorescence of other chromophores and environments, including dyes used to probe the membrane potentials. 

Schummer, U., Schiefer, H. G. and Gerhardt, U. (1979). Mycoplasma membrane-potentials determined by potential-sensitive fluorescent dyes. Curr. Microbiol. 2, 191-194. 

Mitochondria are distinct organelles with two membranes. The outer membrane limits the organelle and the inner membrane is thrown into folds or shelves that project inward and are called cristae mitochondriales. The uptake of most mitochondrion-selective dyes is dependent on the mitochondrial membrane potential. Conventional fluorescent stains for mitochondria, such as rhodamine and tetramethylrosamine, are readily sequestered by functioning mitochondria. They are, however, subsequently washed out of the cells once the mitochondrion s membrane potential is lost. This characteristic limits their use in experiments in which cells must be treated with aldehyde-based fixatives or other agents that affect the energetic state of the mitochondria. To overcome this limitation, the research... 

Presently, the only commercially available dyes that are applied because of then-ability to form fluorescent aggregates are trimethine cyanines JC-1 and JC-9 (Fig. 11) [25], the first one being studied much more extensively than the second one. The dye JC-1 is known to form red-fluorescent (emission maximum at 590 nm) J-aggregates in mitochondria possessing strong intramitochondrial negative potential, while upon depolarization of the mitochondrial membrane, the dye monomer green emission (maximum at 527 nm) is observed [25]. JC-9 demonstrates similar properties [25]. Such properties permit the application of these dyes for, e.g., detection of apoptotic electrical depolarization of mitochondria [25]. 

Mitochondrial function and potential dmg effects such as uncoupling and inhibition of OXPHOS are most directly studied by monitoring oxygen consumption or membrane potential, using polarographic electrodes and fluorescent dyes, respectively, but other techniques are available, as noted below. 

Dykens, J.A. and Stout, A.K. (2001) Fluorescent dyes and assessment of mitochondrial membrane potential in FRET Modes. Fluorescent dyes and assessment of mitochondrial membrane potential in FRET modes. Methods in Cell Biology, 65, 285-309. 

Despite the fact that the fluorescent dyes had many unwanted characteristics, such as interference with mitochondrial ATP production, they were highly useful for the determination of the membrane potential, A /, in cells capable of maintaining ATP levels by glycolytic means. Lipophilic cations have also been used to assess potentials in small cells and permitted concomitant measurement of a potential difference and solute accumulation (Skulachev, 1971 Schuldiner and Kaback, 1975 Lever, 1977). 

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