Slow non-specific accumulation of 2′-deoxy and 2′-O-methyl oligonucleotide probes at mitochondria in live cells

Molecular beacons (MBs) have the potential to provide a powerful tool for rapid RNA detection in living cells, as well as monitoring the dynamics of RNA expression in response to external stimuli. To exploit this potential, it is necessary to distinguish true signal from background signal due to non-specific interactions. Here, we show that, when cyanine-dye labeled 2′-deoxy and 2′-O-methyl oligonucleotide probes are inside living cells for >5 h, most of their signals co-localize with mitochondrial staining. These probes include random-sequence MB, dye-labeled single-strand linear oligonucleotide and dye-labeled double-stranded oligonucleotide. Using carbonyl cyanide m-chlorophenyl hydrazone treatment, we found that the non-specific accumulation of oligonucleotide probes at mitochondria was driven by mitochondrial membrane potential. We further demonstrated that the dye-labeled oligonucleotide probes were likely on/near the surface of mitochondria but not inside mitochondrial inner membrane. Interestingly, oligonucleotides probes labeled respectively with Alexa Fluor 488 and Alexa Fluor 546 did not accumulate at mitochondria, suggesting that the non-specific interaction between dye-labeled ODN probes and mitochondria is dye-specific. These results may help design and optimize fluorescence imaging probes for long-time RNA detection and monitoring in living cells.     

Compared flow cytometric analysis of mitochondria using 10-n-nonyl acridine orange and rhodamine 123

The use of the supravital mitochondrial-specific dye Rhodamine 123 (Rh 123) in combination with flow cytometry permits the monitoring of the changes in the mitochondrial transmembrane potential, reflecting the overall mitochondrial activity of the living cell. While this probe appears to be a potent tool for these studies, it also exhibits an important limit in the interpretation of the results: it cannot distinguish between an increase in mitochondrial activity without biogenesis and a modification of mitochondrial content. 10-n-Nonyl Acridine Orange chloride (NAO) constitutes another mitochondrial specific fluorochrome. In contrast with Rh 123, NAO accumulation in the cell does not seem to be driven by the proton-motrice force but does seem to be related to specific interactions with mitochondrial membrane proteins and/or lipids. In this work, the cytotoxicity of NAO, the kinetics of cellular uptake and the release of the dye have been determined using flow cytometry. The use of several ionophores or mitochondrial inhibitors has confirmed the independence of NAO uptake regarding mitochondrial transmembrane potential. NAO was also used to examine the changes in the mitochondrial compartment during the transfer of articular chondrocytes from cartilage to the culture conditions, where Rh 123 evidenced changes in mitochondrial activity and/or biogenesis, in order to know whether the use of probes with different specificity allows one to distinguish between mitochondrial activity and biogenesis.     

Simultaneous monitoring of ionophore- and inhibitor-mediated plasma and mitochondrial membrane potential changes in cultured neurons

Although natural and synthetic ionophores are widely exploited in cell studies, for example, to influence cytoplasmic free calcium concentrations and to depolarize in situ mitochondria, their inherent lack of membrane selectivity means that they affect the ion permeability of both plasma and mitochondrial membranes. A similar ambiguity affects the interpretation of signals from fluorescent membrane-permeant cations (usually termed "mitochondrial membrane potential indicators"), because the accumulation of these probes is influenced by both plasma and mitochondrial membrane potentials. To resolve some of these problems a technique is developed to allow simultaneous monitoring of plasma and mitochondrial membrane potentials at single-cell resolution using a cationic and anionic fluorescent probe. A computer program is described that transforms the fluorescence changes into dynamic estimates of changes in plasma and mitochondrial potentials. Exploiting this technique, primary cultures of rat cerebellar granule neurons display a concentration-dependent response to ionomycin: low concentrations mimic nigericin by hyperpolarizing the mitochondria while slowly depolarizing the plasma membrane and maintaining a stable elevated cytoplasmic calcium. Higher ionomycin concentrations induce a stochastic failure of calcium homeostasis that precedes both mitochondrial depolarization and an enhanced rate of plasma membrane depolarization. In addition, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone only selectively depolarizes mitochondria at submicromolar concentrations. ATP synthase reversal following respiratory chain inhibition depolarizes the mitochondria by 26 mV.     

Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy

Permeant cationic fluorescent probes are shown to be selectively accumulated by the mitochondria of living cells. Mitochondria-specific interaction of such molecules is apparently dependent on the high trans- membrane potential (inside negative) maintained by functional mitochondria. Dissipation of the mitochondrial trans-membrane and potential by ionophores or inhibitors of electron transport eliminates the selective mitochondrial association of these compounds. The application of such potential-dependent probes in conjunction with fluorescence microscopy allows the monitoring of mitochondrial membrane potential in individual living cells. Marked elevations in mitochondria- associated probe fluorescence have been observed in cells engaged in active movement. This approach to the analysis of mitochondrial membrane potential should be of value in future investigations of the control of energy metabolism and energy requirements of specific biological functions at the cellular level.     

Evaluating Mitochondrial Membrane Potential in Cells

Permeant cationic fluorescent probes are widely employed to monitor mitochondrial transmembrane potential and its changes. The application of such potential-dependent probes in conjunction with both fluorescence microscopy and fluorescence spectroscopy allows the monitoring of mitochondrial membrane potential in individual living cells as well as in large population of cells. These approaches to the analysis of membrane potential is of extremely high value to obtain insights into both the basic energy metabolism and its dysfunction in pathologic cells. However, the use of fluorescent molecules to probe biological phenomena must follow the awareness of some principles of fluorescence emission, quenching, and quantum yield since it is a very sensitive tool, but because of this extremely high sensitivity it is also strongly affected by the environment. In addition, the instruments used to monitor fluorescence and its changes in biological systems have also to be employed with cautions due to technical limits that may affect the signals. We have therefore undertaken to review the most currently used analytical methods, providing a summary of practical tips that should precede data acquisition and subsequent analysis. Furthermore, we discuss the application and feasibility of various techniques and discuss their respective strength and weakness.     

Use of NADH fluorescence to determine mitochondrial function in vivo

Normal mitochondrial function is a critical factor in maintaining cellular homeostasis in various organs of the body. Due to the involvement of mitochondrial dysfunction in many pathological states, the real-time in vivo monitoring of the mitochondrial metabolic state is crucially important. This type of monitoring in animal models as well as in patients provides real-time data that can help interpret experimental results or optimize patient treatment. In this paper we are summarizing the following items: (1) presenting the solid scientific ground underlying nicotine amide adenine dinucleotide (NADH) NADH fluorescence measurements based on published materials. (2) Presenting NADH fluorescence monitoring and its physiological significance. (3) Providing the reader with basic information on the methodologies of the fluorometers reflectometers. (4) Clarifying various factors affecting the monitored signals, including artifacts. (5) Presenting the potential use of monitoring mitochondrial function in vivo for the evaluation of drug development. The large numbers of publications by different groups testify to the valuable information gathered in various experimental conditions. The monitoring of NADH levels in the tissue provides the most important information on the metabolic state of the mitochondria in terms of energy production and intracellular oxygen levels. Although NADH signals are not calibrated in absolute units, their trend monitoring is important for the interpretation of physiological or pathological situations. To better understand the tissue function, the multiparametric approach has been developed where NADH serves as the key parameter to be monitored.     

Staining of mitochondrial membranes with 10-nonyl acridine orange, MitoFluor Green, and MitoTracker Green is affected by mitochondrial membrane potential altering drugs

BACKGROUND: We set out to develop an assay for the simultaneous analysis of mitochondrial membrane potential and mass using the probes 10-nonyl acridine orange (NAO), MitoFluor Green (MFG), and MitoTracker Green (MTG) in HL60 cells. However, in experiments in which NAO and MFG were combined with orange emitting mitochondrial membrane potential (DeltaPsi(m)) probes, we found clear responses to DeltaPsi(m) altering drugs for both probes. METHODS: The three probes were titrated to determine whether saturation played a role in the response to drugs. The effects of a variety of DeltaPsi(m) altering drugs were tested for MFG and MTG at probe concentrations of 20 nM and 200 nM and for NAO at 0.1 microM and 5 microM, using rhodamine 123 at 0.1 microM as a reference probe. RESULTS: Incubation of GM130, HL60, and U937 cells with 2,3-butanedione monoxime (BDM), nigericin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), 2,4-dinitrophenol (DNP), gramicidin, ouabain, and valinomycin resulted in increases of the fluorescence intensity for MFG or MTG with only a few exceptions. The fluorescence intensity of cells stained with 0.1 microM NAO increased following incubation with BDM, nigericin, and decreased for FCCP, CCCP, DNP, gramicidin, and valinomycin. The results with 5 microM NAO were similar. CONCLUSIONS: MFG, MTG, and NAO appeared poor choices for the membrane potential independent analysis of mitochondrial membrane mass. Considering the molecular structure of these probes that favor accumulation in the mitochondrial membrane because of a positive charge, our results are not surprising. Cytometry 39:203-210, 2000. Published 2000 Wiley-Liss, Inc.     

Vitamin E deficiency impairs the modifications of mitochondrial membrane potential and mass in rat splenocytes stimulated to proliferate

This study was designed to evaluate the time-dependent changes of mitochondrial membrane potential and mass during Con-A-induced proliferation of splenic lymphocytes from rat fed a normal or a vitamin E deficient diet. Rhodamine 123 and Nonyl Acridine Orange were used as specific probes to monitor the membrane potential and mass of mitochondria, respectively, by means of flow cytometry. The results demonstrate that the increase of Rh-123 and NAO uptake observed in cells from normally fed rats was prevented by vitamin E deficiency, at any time considered. After 72 h from Con A stimulation, 62% of cells from controls, as against 16% of cells from vitamin E deficient rats, showed hyperpolarized mitochondria. At the same time, in this last group, 60% of cells had depolarized organelles. The same pattern was observed considering the changes of mitochondrial mass, measured using NAO as a probe. These data support that mitogenic stimulation induced an increase of the respiratory activity of mitochondria with subsequent production of superoxide radicals. This resulted in depolarization and loss of mass of the organelles if the intracellular level of vitamin E is not adequate.     

OMA1—An integral membrane protease?

OMA1 is a mitochondrial protease. Among its substrates are DELE1, a signaling peptide, which can elicit the integrated stress response, as well as the membrane-shaping dynamin-related GTPase OPA1, which can drive mitochondrial outer membrane permeabilization. OMA1 is dormant under physiological conditions but rapidly activated upon mitochondrial stress, such as loss of membrane potential or excessive reactive oxygen species. Accordingly, OMA1 was found to be activated in a number of disease conditions, including cancer and neurodegeneration. OMA1 has a predicted transmembrane domain and is believed to be tethered to the mitochondrial inner membrane. Yet, its structure has not been resolved and its context-dependent regulation remains obscure. Here, I review the literature with focus on OMA1's biochemistry. I provide a good homology model of OMA1's active site with a root-mean-square deviation of 0.9 Å and a DALI Z-score of 19.8. And I build a case for OMA1 actually being an integral membrane protease based on OMA1's role in the generation of small signaling peptides, its functional overlap with PARL, and OMA1's homology with ZMPSTE24. The refined understanding of this important enzyme can help with the design of tool compounds and development of chemical probes in the future.     

Mitochondrial membrane potential regulation is independent of c-fos expression.

Tumour cells contain mitochondria with elevated membrane potentials compared with normal cells, and thus this feature provides a selective target for destroying tumour cells. To improve mitochondrial-based therapies, a better understanding of the factors involved in regulating mitochondria are required. Since v-fos overexpression has been shown to elevate mitochondrial membrane potentials in rat fibroblasts, we investigated whether the human homologue, c-fos, was also capable of regulating the mitochondrial membrane potential in cells. Rat fibroblasts transfected with the c-fos gene did not accumulate more rhodamine 123 (Rh123) nor did they retain this Rh123 for extended periods of time compared with their parental line. Moreover, there was no difference in survival following dequalinium chloride (Deca) treatment between transfectants and controls. Similarly, reduction of c-fos expression in rat fibroblasts did not significantly alter their mitochondrial membrane potential. In addition, human ovarian carcinoma cells, which overexpress the c-fos gene, did not accumulate more Rh123 nor were they hypersensitive to Deca compared with their parental line. In another human ovarian carcinoma cell line, selection of variants with lower mitochondrial membrane potential did not alter c-fos mRNA or protein levels. These data suggest that alterations in c-fos expression do not regulate the magnitude of the mitochondrial membrane potential.     

Assessment of sperm damages during different stages of cryopreservation in water buffalo by fluorescent probes

The present study was designed to investigate the sperm damages occurring in acrosome, plasma membrane, mitochondrial activity, and DNA of fresh, equilibrated and frozen–thawed buffalo semen by fluorescent probes. The stability of sperm acrosome and plasma membrane stability, mitochondrial activity and DNA status were assessed by fluorescein conjugated lectin Pisum sativum agglutinin, Annexin–V/propidium iodide, JC-1 and TUNEL assay, respectively, under the fluorescent microscope. The damages percentage of acrosome integrity was significantly increased during equilibration and freezing–thawing process. The stability of sperm plasma membrane is dependent on stability of phosphatidylserine (PS) on the inner leaflet of plasma membrane. The frozen–thawed sperm showed externalization of PS leading to significant increase in apoptotic, early necrotic and necrotic changes and lowered high mitochondrial membrane potential as compared with the fresh sperm but all these parameters were not affected during equilibration. However, the DNA integrity was not affected during equilibration and freezing–thawing procedure. In conclusion, the present study revealed that plasma membrane and mitochondria of buffalo sperm are more susceptible to damage during cryopreservation. Furthermore, the use of fluorescent probes to evaluate integrity of plasma and acrosome membranes, as well as mitochondrial membrane potential and DNA status increased the accuracy of semen analyses.     

Mitochondrial membrane potential selects hybridomas yielding high viability in fed-batch cultures

Prior research (Follstad, B. D.; Wang, D. I. C.; Stephanopoulos, G. Mitochondrial membrane potential differentiates cells resistant to apoptosis in hybridoma cultures. Eur. J. Biochem. 2000, 267, 6534-6540.) identified mitochondrial membrane potential (MMP) as a marker of hybridoma subpopulations resistant to apoptosis caused by a variety of apoptosis inducers. In this study, we investigated the viability of hybridoma cell cultures inoculated with cells of varying MMP in regular fed-batch operation. A hybridoma cell population was separated using FACS into subpopulations based on their mean mitochondrial membrane potential (MMP) as measured using the common mitochondrial stain, Rhodamine 123 (Rh123). These subpopulations showed dramatic differences in their apoptotic death kinetics. Fed-batches inoculated with a high MMP subpopulation reached higher viable cell concentrations and viabilities that were maintained for prolonged periods of time relative to fed-batches inoculated with low MMP subpopulations. These results underline the heterogeneous nature of hybridoma cell cultures and suggest that mitochondrial physiology is a critical parameter determining culture performance.     

Coenzyme Q10 Protects Astrocytes from ROS-Induced Damage through Inhibition of Mitochondria-Mediated Cell Death Pathway

Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species to protect neuronal cells against oxidative stress in neurodegenerative diseases. The present study was designed to examine whether CoQ10 was capable of protecting astrocytes from reactive oxygen species (ROS) mediated damage. For this purpose, ultraviolet B (UVB) irradiation was used as a tool to induce ROS stress to cultured astrocytes. The cells were treated with 10 and 25 μg/ml of CoQ10 for 3 or 24 h prior to the cells being exposed to UVB irradiation and maintained for 24 h post UVB exposure. Cell viability was assessed by MTT conversion assay. Mitochondrial respiration was assessed by respirometer. While superoxide production and mitochondrial membrane potential were measured using fluorescent probes, levels of cytochrome C (cyto-c), cleaved caspase-9, and caspase-8 were detected using Western blotting and/or immunocytochemistry. The results showed that UVB irradiation decreased cell viability and this damaging effect was associated with superoxide accumulation, mitochondrial membrane potential hyperpolarization, mitochondrial respiration suppression, cyto-c release, and the activation of both caspase-9 and -8. Treatment with CoQ10 at two different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide, normalization of mitochondrial membrane potential, improvement of mitochondrial respiration, inhibition of cyto-c release, suppression of caspase-9. Furthermore, CoQ10 enhanced mitochondrial biogenesis. It is concluded that CoQ10 may protect astrocytes through suppression of oxidative stress, prevention of mitochondrial dysfunction, blockade of mitochondria-mediated cell death pathway, and enhancement of mitochondrial biogenesis.     

Cooperation of phosphatidylcholine-specific phospholipase C and basic fibroblast growth factor in the neural differentiation of mesenchymal stem cells in vitro

Previously, we found that suppressing phosphatidylcholine-specific phospholipase C could induce neuronal differentiation of rat mesenchymal stem cells in the absence of serum and fibroblast growth factor. It is well known that basic fibroblast growth factor plays an important role in mesenchymal stem cell neuronal differentiation. In this study, our purpose was to understand the cooperation of phosphatidylcholine-specific phospholipase C and basic fibroblast growth factor in controlling mesenchymal stem cell neuronal differentiation. Our results showed that suppressing phosphatidylcholine-specific phospholipase C in the presence of basic fibroblast growth factor could induce cell neuronal differentiation and the viability of the differentiated cells was obviously increased. Furthermore, we found that the resting membrane potential of the differentiated cells gradually decreased, but the mitochondrial membrane potential rose with increasing treatment time and these characteristics were similar to cultured neurons from mouse embryo forebrains. To determine the possible mechanism by which this combination controls cell neuronal differentiation, we measured changes in the mitochondrial membrane potential and in the levels of reactive oxygen species. The results showed that both the mitochondrial membrane potential and reactive oxygen species levels decreased when basic fibroblast growth factor was added. The data suggested that lower phosphatidylcholine-specific phospholipase C activity was required for mesenchymal stem cell neuronal differentiation and basic fibroblast growth factor was necessary for maintaining the neuronal differentiation state. Moreover, basic fibroblast growth factor could contribute to rescuing the differentiated cells from death through decreasing overly high mitochondrial membrane potentials and reactive oxygen species levels.     

ABC transporters affect the detection of intracellular oxidants by fluorescent probes

Intracellular production of reactive oxygen species (ROS) plays an important role in the control of cell physiology. For the assessment of intracellular ROS production, a plethora of fluorescent probes is commonly used. Interestingly, chemical structures of these probes imply they could be substrates of plasma membrane efflux pumps, called ABC transporters. This study tested whether the determination of intracellular ROS production and mitochondrial membrane potential by selected fluorescent probes is modulated by the expression and activity of ABC transporters. The sub-clones of the HL-60 cell line over-expressing MDR1, MRP1 and BCRP transporters were employed. ROS production measured by luminol- and L-012-enhaced chemiluminescence and cytochrome c reduction assay showed similar levels of ROS production in all the employed cell lines. It was proved that dihydrorhodamine 123, dihexiloxocarbocyanine iodide, hydroethidine, tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide and tetramethylrhodamine ethyl ester perchlorate are substrates for MDR1; dichlorodihydrofluoresceine, hydroethidine and tetramethylrhodamine ethyl ester perchlorate are substrates for MRP1; dichlorodihydrofluoresceine, dihydrorhodamine 123, hydroethidine and tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide are substrates for BCRP. Thus, the determination of intracellular ROS and mitochondrial potential by the selected probes is significantly altered by ABC transporter activities. The activity of these transporters must be considered when employing fluorescent probes for the assessment of ROS production or mitochondrial membrane potential.     

Inducible proteolytic inactivation of OPA1 mediated by the OMA1 protease in mammalian cells

The mammalian mitochondrial inner membrane fusion protein OPA1 is controlled by complex patterns of alternative splicing and proteolysis. A subset of OPA1 isoforms is constitutively cleaved by YME1L. Other isoforms are not cleaved by YME1L, but they are cleaved when mitochondria lose membrane potential or adenosine triphosphate. In this study, we show that this inducible cleavage is mediated by a zinc metalloprotease called OMA1. We find that OMA1 small interfering RNA inhibits inducible cleavage, helps retain fusion competence, and slows the onset of apoptosis, showing that OMA1 controls OPA1 cleavage and function. We also find that OMA1 is normally cleaved from 60 to 40 kD by another as of yet unidentified protease. Loss of membrane potential causes 60-kD protein to accumulate, suggesting that OMA1 is attenuated by proteolytic degradation. We conclude that a proteolytic cascade controls OPA1. Inducible cleavage provides a mechanism for quality control because proteolytic inactivation of OPA1 promotes selective removal of defective mitochondrial fragments by preventing their fusion with the mitochondrial network.     

Mitochondrial Membrane Studies Using Impedance Spectroscopy with Parallel pH Monitoring

A biological microelectromechanical system (BioMEMS) device was designed to study complementary mitochondrial parameters important in mitochondrial dysfunction studies. Mitochondrial dysfunction has been linked to many diseases, including diabetes, obesity, heart failure and aging, as these organelles play a critical role in energy generation, cell signaling and apoptosis. The synthesis of ATP is driven by the electrical potential across the inner mitochondrial membrane and by the pH difference due to proton flux across it. We have developed a tool to study the ionic activity of the mitochondria in parallel with dielectric measurements (impedance spectroscopy) to gain a better understanding of the properties of the mitochondrial membrane. This BioMEMS chip includes: 1) electrodes for impedance studies of mitochondria designed as two- and four-probe structures for optimized operation over a wide frequency range and 2) ion-sensitive field effect transistors for proton studies of the electron transport chain and for possible monitoring other ions such as sodium, potassium and calcium. We have used uncouplers to depolarize the mitochondrial membrane and disrupt the ionic balance. Dielectric spectroscopy responded with a corresponding increase in impedance values pointing at changes in mitochondrial membrane potential. An electrical model was used to describe mitochondrial sample’s complex impedance frequency dependencies and the contribution of the membrane to overall impedance changes. The results prove that dielectric spectroscopy can be used as a tool for membrane potential studies. It can be concluded that studies of the electrochemical parameters associated with mitochondrial bioenergetics may render significant information on various abnormalities attributable to these organelles.     

Doxorubicin induces mitochondrial permeability transition and contractile dysfunction in the human myocardium

In human atrial trabeculae, we examined the effects of doxorubicin on the isometric force of contraction, mitochondrial respiration, membrane potential and calcium retention capacity. Compared with untreated controls, doxorubicin induced contractile dysfunction and depression of mitochondrial respiration. Mitochondria isolated from doxorubicin-treated human atrial trabeculae displayed reduced transmembrane potential and calcium retention capacity. Cyclosporine A, a mitochondrial membrane transition pore opening blocker, prevented mitochondrial dysfunction and impaired contractile performance induced by doxorubicin. The study suggests that a mitochondrial membrane transition pore opening is involved in the development of doxorubicin cardiotoxicity in human hearts.     

Effects of acute leptin administration on the differences in proton leak rate in liver mitochondria from ob/ob mice compared to lean controls.

In this investigation, the effects on proton leak of leptin administration to ob/ob mice was measured for liver mitochondria. We and others have shown that proton leak is approximately 3 times greater in liver mitochondria from ob/ob mice compared to lean controls at any given membrane potential. The results are consistent with obese mammals having higher lean mass-specific metabolic rates compared to lean controls. The increase in proton leak rate at any given membrane potential cannot be explained by the presence of free fatty acids associated with mitochondria isolated from the obese animals. The difference in proton leak must therefore represent a real difference in inner membrane permeability. Acute leptin (OB protein) administration restores the liver mitochondrial proton leak rate of ob/ob mice to that of lean controls. There was no effect on proton leak rate of liver mitochondria from sham-treated ob/ob mice. These novel results indicate a role for leptin, either directly or indirectly, in regulating the efficiency of oxidative phosphorylation.     

Dicarbocyanine fluorescent probes of membrane potential block lymphocyte capping, deplete cellular ATP and inhibit respiration of isolated mitochondria.

3,3'-Dipropylthiodicarbocyanine iodide, a widely used fluorescent probe of membrane potential, was found to inhibit anti-Ig antibody, induced capping of mouse lymphocytes. The dye also lowered the cell ATP content. Experiments with isolated mitochondria revealed that the probe had a potent inhibitory action at site I of the respiratory chain. This mitochondrial blockade helps to explain the ATP depletion and blockade of capping, and gives cause for caution in the use of this dye as a probe of cell membrane potential. Three related dicarbocyanine dyes had similar toxic effects, but two cyanine dyes with much longer alkyl side chains, which have been used as probes of membrane fluidity, did not.