The use of flurescent dyes to measure membrane potentials: A response

Smith, Thomas C.

doi:10.1002/jcp.1041120222

Cited by 27 publications

(12 citation statements)

References 21 publications

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“…Johnstone et al, 1982;Smith 1982). Estimates of V,,+ based on indirect methods generally give values greater than those obtained by direct electrophysiological techniques (Laris et al, 1976, Philo & Eddy, 1978Smith & Robinson, 1981b).…”

Section: Discussionmentioning

confidence: 97%

“…In general, two different methods have been applied to estimate Vm: direct electrophysiological measurements utilizing intracellular microelectrodes, and indirect determinations utilizing passively distributed ions. The estimates achieved by these approaches are not always in good agreement (@ Johnstone, Laris & Eddy, 1982;Smith, 1982). Typically, direct measurements by microelectrodes give vahles in the range -20 to -30 mV (Lassen et al, 1971;Smith & Vernon, 1979;Smith & Robinson, 1981b;Dawson & Smith, 1986), while indirect estimates are significantly more elevated (-40 to -60 mV; Laris et al, 1976;Hoffmann & Lambert, 1983;Valdeolmillos, GarciaSancho & Herreros, 1986).…”

Section: Introductionmentioning

confidence: 93%

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Validation of the use of the lipophilic thiocyanate anion for the determination of membrane potential in Ehrlich ascites tumor cells

Smith

Robinson

1989

J. Membrain Biol.

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The utility of the lipophilic anion thiocyanate (SCN-) as a probe for the indirect estimation of the cell membrane potential (Vm) in Ehrlich ascites tumor cells has been evaluated by comparison to direct electrophysiological measurements. SCN accumulation is consistent with first-order uptake into a single, kinetically-identifiable cellular compartment, achieving steady-state distribution in 20-30 min at 22 degrees C. The steady state distribution ratio ([SCN-]e/[SCN-]e) in physiological saline is 0.44 +/- 0.02. Treatment of the cells with propranolol (0.13 mM), an activator of Ca2+ dependent K+ channels, reduces the steady-state distribution ratio to 0.19 +/- 0.02. Conversely, treatment with BaCl2 (10 mM), an antagonist of the pathway, increases the SCN- distribution ratio to 0.62 +/- 0.01. The equilibrium potentials (VSCN) calculated under these conditions are virtually identical to direct electrophysiological measurements of the Vm made under the same conditions. The effect of varying extracellular [K+] ([K+]e) in the presence of constant [Na+]e = 100 mM has also been tested. In control cells, elevation of [K+]e from 6 to 60 mM reduces VSCN from -20.6 +/- 1.0 to -13.2 +/- 1.2 mV. Again, microelectrode measurements give excellent quantitative agreement. Propranolol increases the sensitivity of the cells to varying [K+]e, so that a 10-fold elevation reduces VSCN by approximately 31 mV. BaCl2 greatly reduces this response: a 10-fold elevation in [K+]e yielding only a 4-mV reduction in VSCN. It is concluded that the membrane potential of Ehrlich cells can be estimated accurately from SCN- distribution measurements.

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Section: Discussionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 93%

Validation of the use of the lipophilic thiocyanate anion for the determination of membrane potential in Ehrlich ascites tumor cells

Smith

Robinson

1989

J. Membrain Biol.

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“…Several side effects of the dye have, however, been under discussion (for example, see Johnstone et al, 1982;Smith, 1982). The major points investigated have been, the effect of the dye-induced inhibition of the respiration, and an eventual change in the K + permeability caused by the dye.…”

Section: Evaluation Of the Fluorescense Methods And The Thiocyanate DImentioning

confidence: 99%

“…Where most of the microelectrode measurements may have given erroneously low values for the membrane potentials due to leakage caused by the microelectrode impalements, the results obtained with the dyes have, on the other hand, been questioned for the possible effects of the dye on the ionic conductances as well as errors introduced by the calibration of the dyes using the "valinomycin null-point" method (see Johnstone, Laris & Eddy, 1982;Smith, 1982). In the present report, the fluorescence of the dye DiOC3-(5) is calibrated in Na+-free media using gramicidin instead of valinomycin to increase the cation permeability of the cell membrane.…”

Section: Introductionmentioning

confidence: 99%

Membrane potential, anion and cation conductances in Ehrlich ascites tumor cell

Lambert

Hoffmann

Jørgensen³

1989

J. Membrain Biol.

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The fluorescence intensity of the dye 1,1'-dipropylox-adicarbocyanine (DiOC3-(5] has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (Vm) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na+-free K-/choline-media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155 mM. Calibration by the valinomycin "null point" procedure described by Laris et al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976, Biochim, Biophys. Acta 436:475-488) is shown to be valid only in the presence of the Cl- -channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN- as an indirect estimation of the membrane potential is found not to be applicable for the fast changes in Vm reported in this paper. Incubation with DiOC3-(5) for 5 min is demonstrated to reduce the Cl permeability by 26 +/- 5% and the NO3- permeability by 15 +/- 2%, while no significant effect of the probe could be demonstrated on the K+ permeability. Values for Vm, corrected for the inhibitory effect of the dye on the anion conductance, are estimated at -61 +/- 1 mV in isotonic standard NaCl medium, -78 +/- 3 mV in isotonic Na+-free choline medium and -46 +/- 1 mV in isotonic NaNO3 medium. The cell membrane is depolarized by addition of the K+ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na+-free choline medium, indicating that Vm is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO3- for all cellular and extracellular Cl- leads to a depolarization of the membrane, demonstrating that Vm is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K+ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 microS/cm2 for K+, 3.0 microS/cm2 for Na+, 0.6 microS/cm2 for Cl- and 8.7 microS/cm2 for NO3-. Addition of the Ca2+-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K+ permeability exceeds the Cl- permeability also after the addition of A23187. The K+ and Cl- conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 microS/cm2, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

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“…Because mitochondrial dye binding and fluorescence eficiency are not constant, calibration methods (e.g., valinomycin) that depolarize the mitochondria induce a complex bias into the calculation of qpIp in intact cells. A number of other potential pitfalls in ionophore calibration have been identified (Smith, 1982;Azzone et al, 1984). The conditions that we find to optimize the P signal, i.e., high dye/cell ratios, prolong dye equili&yium time (Zanotti and Azzone, 19801, particularly the equilibrium between the medium and the fluidics tubing, and increase the likelihood of dye both introducing a significant extrinsic permeability and participating in gradient-altering probe-cation exchange reactions (Bashford and Smith, 1979).…”

Section: (5)mentioning

confidence: 97%

Voltage‐sensitive cyanine dye fluorescence signals in lymphocytes: Plasma membrane and mitochondrial components

Wilson

Seligmann

Chused

1985

Journal Cellular Physiology

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The origin of the cyanine dye fluorescence signal in murine and human peripheral blood leukocytes was investigated using the oxa- and indo-carbocyanines di-O-C5(3) and di-I-C5(3). Fluorescence signals from individual cells suspended with nanomolar concentrations of the dyes were measured in a flow cytometer modified to permit simultaneous four-parameter analysis (including two-color fluorescence or fluorescence polarization measurements). The contributions of mitochondrial membrane potential (psi m) and plasma membrane potential (psi pm) to the total voltage-sensitive fluorescence signal were found to depend on the equilibrium extracellular dye concentration, manipulated in these experiments by varying the ratio of dye to cell density. Hence, conditions could be chosen that amplified either the psi m or the psi pm component. Selective depolarization of lymphocytes or polymorphonuclear leukocytes (PMN) in mixed cell suspensions demonstrated that defining the partition of dye between cells and medium is requisite to assessing the heterogeneity of cell responses by cyanine dye fluorescence. At extracellular dye concentrations exceeding 5 nM in equilibrated cell suspensions, both mitochondrial and plasma membrane dye toxicity were observed. In murine splenic lymphocytes, plasma membrane toxicity (dye-induced depolarization) was selective for the B lymphocytes. Certain problems in calibration of psi pm with valinomycin at low dye concentrations and perturbations of psi pm by mitochondrial inhibitors are presented. These findings address the current controversy concerning psi m and psi pm measurement in intact cells by cyanine dye fluorescence. The finding of selective toxicity at low cyanine dye concentrations suggest that purported differences in resting psi m among cells or changes in psi pm with cell activation may reflect variable susceptibility to dye toxicity rather than intrinsic cell properties.

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The use of flurescent dyes to measure membrane potentials: A response

Cited by 27 publications

References 21 publications

Validation of the use of the lipophilic thiocyanate anion for the determination of membrane potential in Ehrlich ascites tumor cells

Validation of the use of the lipophilic thiocyanate anion for the determination of membrane potential in Ehrlich ascites tumor cells

Membrane potential, anion and cation conductances in Ehrlich ascites tumor cell

Voltage‐sensitive cyanine dye fluorescence signals in lymphocytes: Plasma membrane and mitochondrial components

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