Subject(s)
Membrane Potentials , Saccharomyces cerevisiae/physiology , Spectrometry, Fluorescence/methods , Aerobiosis , Anaerobiosis , Benzothiazoles , Carbocyanines , Fluorescent Dyes , Glucose/metabolism , Potassium/pharmacology , Saccharomyces cerevisiae/drug effects , Uncoupling Agents/pharmacologyABSTRACT
Three fluorescent probes, tetramethyl rhodamine ethyl ester (TMRE), 3,3'-dipropylthiacarbocyanine iodide (diS-C3(3)) and 3,3'-dipropyloxacarbocyanine iodide (diO-C3(3)), were tested for their suitability as fluorescent indicators of membrane potential in Saccharomyces cerevisiae in studies performed by flow cytometry. For all these dyes the intensity of fluorescence of stained cells increased with probe concentration in the range of 60-3000 nmol/L. The optimum staining period was 15-20 min for diS-C3(3). Depolarization of cells by increased extracellular potassium level and by valinomycin elicited with all probes a drop in fluorescence intensity. In some yeast batches this depolarization was accompanied by a separation of subpopulations with different fluorescence properties.
Subject(s)
Carbocyanines , Flow Cytometry/methods , Fluorescent Dyes , Fluorometry , Membrane Potentials , Saccharomyces cerevisiae/physiology , Artifacts , Benzothiazoles , Fluorescence , Indicators and Reagents , Organometallic Compounds , Pyridinium Compounds/analysis , Pyridinium Compounds/metabolism , Scattering, Radiation , Time FactorsABSTRACT
Some slow potentiometric dyes, e.g., 3,3'-dipropylthiacarbocyanine and tetramethylrhodamine methyl ester, exhibit fluorescence spectral changes on redistributing from aqueous medium to cells. This effect has been used for spectroscopic discrimination of the emissions from free and bound dyes in cells. Such a discrimination can, in principle, allow for the assessment of cell membrane potential in individual cells, using the Nernst equation applied to the ratio of free dye fluorescence intensities inside and outside of the cell.
