Transmembrane potentials in cells: a diS-C3(3) assay for relative potentials as an indicator of real changes.

2.50
Hdl Handle:
http://hdl.handle.net/10541/96101
Title:
Transmembrane potentials in cells: a diS-C3(3) assay for relative potentials as an indicator of real changes.
Authors:
Plásek, J; Dale, Robert E; Sigler, K; Laskay, Gabor
Abstract:
The mechanism by which the fluorescent cationic dye diS-C3(3) reports on cellular transmembrane potential has been investigated in murine haemopoietic cells. Due to the large molar absorbance of diS-C3(3) and its high quantum yield of fluorescence in cells, this dye can be used at very low labelling concentrations (5 x 10(-8) to 2 x 10(-7) M). In contrast to the quenching of fluorescence observed for the most commonly used voltage-sensitive dyes of the carbocyanine class, the fluorescence intensity of diS-C3(3) increases when the dye accumulates in the cells. The method of synchronous emission spectroscopy was used to resolve the intracellular and extracellular components of the diS-C3(3) fluorescence of suspensions of labelled cells. In comparing changes in these signals consequent on changes in transmembrane potential induced by varying the extracellular concentration of potassium ions in the presence of valinomycin, the logarithm of the ratio of intensities of these two components, as predicted theoretically, was found to be a good linear measure of transmembrane potential under these conditions. The dye was also demonstrated to be suitable for flow-cytofluorimetric analysis, the logarithm of the mean population signal similarly being found to provide a good linear measure of the transmembrane potential. The conditions under which such linearity may be expected with respect to possible effects due to changes in the capacity for binding of the dye to proteins and various cytosolic structures are delineated and their validity with respect to the possibly contentious role of mitochondria in such measurements examined in particular. The use of the method in indicating changes in the transmembrane potential and/or changes in the transport numbers of the major ions determining transmembrane potential between different physiological states, the possible extension to determinations of absolute differences in potential between different cell states without calibration or comparison with potassium-ion potentials, and the conditions for validity and limitations of these partially complementary measurements, are discussed.
Affiliation:
Institute of Physics of Charles University, Prague, Czech Republic.
Citation:
Transmembrane potentials in cells: a diS-C3(3) assay for relative potentials as an indicator of real changes. 1994, 1196 (2):181-90 Biochim. Biophys. Acta
Journal:
Biochimica et Biophysica Acta
Issue Date:
30-Dec-1994
URI:
http://hdl.handle.net/10541/96101
DOI:
10.1016/0005-2736(94)00209-6
PubMed ID:
7841182
Type:
Article
Language:
en
ISSN:
0006-3002
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorPlásek, Jen
dc.contributor.authorDale, Robert Een
dc.contributor.authorSigler, Ken
dc.contributor.authorLaskay, Gaboren
dc.date.accessioned2010-04-09T08:53:50Z-
dc.date.available2010-04-09T08:53:50Z-
dc.date.issued1994-12-30-
dc.identifier.citationTransmembrane potentials in cells: a diS-C3(3) assay for relative potentials as an indicator of real changes. 1994, 1196 (2):181-90 Biochim. Biophys. Actaen
dc.identifier.issn0006-3002-
dc.identifier.pmid7841182-
dc.identifier.doi10.1016/0005-2736(94)00209-6-
dc.identifier.urihttp://hdl.handle.net/10541/96101-
dc.description.abstractThe mechanism by which the fluorescent cationic dye diS-C3(3) reports on cellular transmembrane potential has been investigated in murine haemopoietic cells. Due to the large molar absorbance of diS-C3(3) and its high quantum yield of fluorescence in cells, this dye can be used at very low labelling concentrations (5 x 10(-8) to 2 x 10(-7) M). In contrast to the quenching of fluorescence observed for the most commonly used voltage-sensitive dyes of the carbocyanine class, the fluorescence intensity of diS-C3(3) increases when the dye accumulates in the cells. The method of synchronous emission spectroscopy was used to resolve the intracellular and extracellular components of the diS-C3(3) fluorescence of suspensions of labelled cells. In comparing changes in these signals consequent on changes in transmembrane potential induced by varying the extracellular concentration of potassium ions in the presence of valinomycin, the logarithm of the ratio of intensities of these two components, as predicted theoretically, was found to be a good linear measure of transmembrane potential under these conditions. The dye was also demonstrated to be suitable for flow-cytofluorimetric analysis, the logarithm of the mean population signal similarly being found to provide a good linear measure of the transmembrane potential. The conditions under which such linearity may be expected with respect to possible effects due to changes in the capacity for binding of the dye to proteins and various cytosolic structures are delineated and their validity with respect to the possibly contentious role of mitochondria in such measurements examined in particular. The use of the method in indicating changes in the transmembrane potential and/or changes in the transport numbers of the major ions determining transmembrane potential between different physiological states, the possible extension to determinations of absolute differences in potential between different cell states without calibration or comparison with potassium-ion potentials, and the conditions for validity and limitations of these partially complementary measurements, are discussed.en
dc.language.isoenen
dc.subject.meshAnimals-
dc.subject.meshCarbocyanines-
dc.subject.meshCell Line-
dc.subject.meshCell Membrane-
dc.subject.meshFluorescent Dyes-
dc.subject.meshMembrane Potentials-
dc.subject.meshMice-
dc.subject.meshMitochondria-
dc.subject.meshSpectrometry, Fluorescence-
dc.titleTransmembrane potentials in cells: a diS-C3(3) assay for relative potentials as an indicator of real changes.en
dc.typeArticleen
dc.contributor.departmentInstitute of Physics of Charles University, Prague, Czech Republic.en
dc.identifier.journalBiochimica et Biophysica Actaen
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