3-3--dipropyloxadicarbocyanine and 3-3--dipropyl-2-2--thiadicarbocyanine

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.

Topics: Artifacts; Benzothiazoles; Carbocyanines; Flow Cytometry; Fluorescence; Fluorescent Dyes; Fluorometry; Indicators and Reagents; Membrane Potentials; Organometallic Compounds; Pyridinium Compounds; Saccharomyces cerevisiae; Scattering, Radiation; Time Factors

The interaction of thymocyte mitochondria with two types of dyes - potential indicators commonly used in lymphocyte studies, has been investigated. Ethylrhodamine at concentrations up to 16 microM does not influence the systems of oxidation and energy coupling in lymphocyte mitochondria. Carbocyanines-diS-C3-(5) and diO-C3-(5) inhibit oxygen uptake by the lymphocytes in the presence of glucose and pyruvate at the same low concentrations as does rotenone (40% inhibition occurs at 10 nM). DNP reduces the inhibition of respiration by carbocyanines but not by rotenone. The increase in the fluorescence of diS-C3-(5) and in the rate of oxygen uptake in the absence of diS-C3-(5) occurs at close concentrations of the uncoupler. This indicates that the changes in the fluorescence caused by FCCP reflect the membrane potential of lymphocyte mitochondria. The maintenance of the membrane potential in lymphocyte mitochondria in the presence of diS-C3-(5) provides evidence for the absence of the corresponding changes in mitochondrial ultrastructure after addition of 0.6 microM diS-C3-(5) which completely inhibits oxygen uptake.

Topics: Animals; Benzothiazoles; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Fluorescent Dyes; Intracellular Membranes; Lymphocytes; Membrane Potentials; Mitochondria; Oxygen Consumption; Quinolines; Rats; Rhodamines; Thymus Gland; Xanthenes

The experiments presented below compare the interaction of diO-C3-(5) and diS-C3-(5) with erythrocytes, erythrocyte ghosts and phospholipid vesicles derived from erythrocyte membranes. The results confirm earlier reports of diS-C3-(5) dimerization in the presence of hemoglobin and of dye aggregate formation in erythrocyte suspensions. DiO-C3-(5), on the other hand, binds to vesicles and ghosts freed of hemoglobin in a potential-dependent manner but without forming dye aggregates. The two dyes bind to the different preparations in similar proportions, but diS-C3-(5) is bound in amounts 3-40 times greater depending on the degree of polarization. The results show that mechanism other than binding to hemoglobin must occur in order to explain the potential-dependent binding of both dyes to ghosts and vesicles. A primary interaction must exist between the dye molecule and the lipid bilayer in a biological membrane, and this would be expected to occur in the presence of hemoglobin or other cytosolic components. DiO-C3-(5) is a better dye to use than diS-C3-(5) for mechanistic studies, in order to avoid problems associated with formation of complex aggregates of the latter dye, especially in hyperpolarized membrane suspensions.

Topics: Benzothiazoles; Carbocyanines; Erythrocyte Membrane; Erythrocytes; Fluorescence; Fluorescent Dyes; Humans; Lipid Bilayers; Membrane Potentials; Potassium; Quinolines; Spectrophotometry