The fluorescence of the voltage sensitive dye, diS-C3-(5), has been analyzed by means of synchronous excitation spectroscopy. Using this rather rare fluorescence technique we have been able to distinguish between the slightly shifted spectra of diS-C3-(5) fluorescence from cells and from the supernatant. It has been found that diS-C3-(5) fluorescence in the supernatant can be selectively monitored at λexc = 630 nm and λem= 650 nm, while the cell associated fluorescence can be observed at λexc= 690 nm and λem = 710 nm. A modified theory for the diSC3-(5) fluorescence response to the membrane potential is presented, according to which a linear relationship exists between the logarithmic increment of the dye fluorescence intensity in the supernatant, In I/I°, and the underlying change in the plasma membrane potential, Δψp=ψp-ψ°p. The theory has been tested on human myeloid leukemia cells (line ML-1) in which membrane potential changes were induced by valinomycin clamping in various K+ gradients. It has been demonstrated that the membrane potential change, Δψp,can be measured on an absolute scale.
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Plášek, J., Hrouda, V. Assessment of membrane potential changes using the carbocyanine dye, diS-C3-(5): synchronous excitation spectroscopy studies. Eur Biophys J 19, 183–188 (1991). https://doi.org/10.1007/BF00196344
- Membrane potential
- Synchronous excitation spectroscopy