Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes

Abstract

The distribution of a selection of cationic fluorescent dyes can be used to measure the membrane potential of individual cells with a microfluorometer. The essential attributes of these dyes include membrane permeability, low membrane binding, spectral properties which are insensitive to environment, and, of course, strong fluorescence. A series of dyes were screened on HeLa cells for their ability to meet these criteria and several commercially available dyes were found to be satisfactory. In addition, two new dyes were synthesized for this work by esterification of tetramethyl rhodamine. The analysis of the measured fluorescent intensities requires correction for fluorescence collected from outside the plane of focus of the cell and for nonpotentiometric binding of the dye. The measurements and analysis were performed on three different cell types for which there exists a body of literature on membrane potential; the potentials determined in this work were always within the range of literature values. The rhodamine esters are nontoxic, highly fluorescent dyes which do not form aggregates or display binding-dependent changes in fluorescence efficiency. Thus, their reversible accumulation is quantitatively related to the contrast between intracellular and extracellular fluorescence and allows membrane potentials in individual cells to be continuously monitored.

Mitochondrial free calcium transients during excitation‐contraction coupling in rabbit cardiac myocytes

“…chondrial compartments, as shown previously [27]. TMRM is a cationic fluorophore that accumulates electrophoretically into mitochondria in response to their highly negative membrane potential [27,29]. In our studies here, we used TMRM simply to identify the mitochondrial compartment.…”

Induction of Mitochondrial Dysfunction by Poly(ADP-Ribose) Polymer: Implication for Neuronal Cell Death

“…In some cases, fluorescent probes intended to monitor mitochondrial functional parameters exhibit nonspecific action depending on doses and durations, or stain non-target cellular organelles other than mitochondria. These artifacts may lead to false interpretation (Ehrenberg et al, 1988;Farkas et al, 1989). To avoid these issues, we used various fluorescent probes that are supposed to have different actions including JC-1, TMRE, MitoTracker Red and calcein in combination with appropriate controls (ionomycin or CCCP).…”

“…CCCP, an uncoupler of oxidative phosphorylation, was used as a positive control for depolarization of mitochondrial potential and cells were treated for 30 min before staining. TMRE is a potential sensitive, cell-permeant fluorescent dye that is readily sequestered by active mitochondria (Ehrenberg et al, 1988;Farkas et al, 1989). Because it is rapidly and reversibly taken up by intact mitochondria in a potential-dependent manner, TMRE has been described as one of the best fluorescent dyes for dynamic and in situ quantitative measurements of mitochondrial potential by means of imaging or flow cytometric analysis (Umegaki et al, 2008).…”

Dynamics of Intracellular Oxygen in PC12 Cells upon Stimulation of Neurotransmission

“…Single Cell Characterization of ⌬⌿ m and ⌬⌿ p Relative to O 2 Consumption Following K ϩ Stimulation-The potentiometric probe TMRM has been used extensively as a tool for the single cell characterization of ⌬⌿ m (36,37,(43)(44)(45)(46) in vitro. Here we have utilized a nonquenching concentration of TMRM (20 nM) to monitor mitochondrial bioenergetics in d PC12 cells relative to changes in i O 2 .…”