Methods of registering trans-membrane electric potentials

Sensitive methods for identifying compounds having biological activity comprising combining living cells with two fluorescent membrane permeable ionic dyes having the same charge sign, the first of which has an emission spectrum which overlaps the excitation spectrum of the second fluorescent membrane penetrative dye. The fluorescence is then induced by illuminating the dyes at a wavelength corresponding to the excitation spectrum of the first fluorescent dye and emission is then registered at a wavelength corresponding to the emission spectrum of the second fluorescent dye (FRET). The change in the FRET is indicative of a modulation of cell membrane potential by the biologically active compounds.

FIELD OF THE INVENTION
 The present invention relates to the field of cell biology, more
 particularly to measuring electric potentials across cell plasma and
 mitochondrial membranes.
 BACKGROUND OF THE INVENTION
 The existence of an electric potential across cell membranes, such as
 plasma membranes or the mitochondria, is a major factor in proper cell
 functioning. Excitatory cells, including neurons or muscle cells, actively
 separate negatively charged molecules inside the cell from positively
 charged ones in the space outside the membrane. This charge distribution
 maintains a steady-state homeostasis trans-membrane electric potential
 that is characteristic for resting cells. Upon cell activation, either
 with an electric field or with specific signal transducing molecules, the
 resting membrane potential changes. This change causes a cell reaction,
 which leads to, for example, neuronal signal propagation or muscular
 contraction.
 The ability to monitor the cell membrane potential in single cells or in
 cell populations is important for understanding both the intricate
 molecular mechanisms underlying cell functioning, as well as for drug
 development. Two main approaches have been developed to monitor cell
 membrane potentials: direct electrical measurements with micro-electrodes
 and indirect measurement of membrane potential by following the
 redistribution of specially developed lipophilic ions labeled with either
 an isotope or a fluorescent moiety.
 Both membrane permeable probes and non-membrane-permeable probes are used
 to monitor cell membrane potentials. Permeable fluorescent probes usually
 have high sensitivity to membrane potential changes but are very slow to
 respond to these changes. Non-permeable probes usually react quickly to
 membrane potential changes but have very low sensitivity. The main
 disadvantage of these probes is that their fluorescence intensity changes
 upon membrane depolarization or hyperpolarization. An approach based on
 fluorescence intensity can be confounded by variations in dye loading,
 cell density, or variability in excitation intensity. Also, it can be
 misleading when one considers the use of compounds with inherent spectral
 characteristics that interfere with the fluorescence of a probe.
 As an example of membrane permeable probes, the class of negatively charged
 oxonols is best suited for measuring plasma membrane potentials because
 they are excluded from entering mitochondria due to the high negative
 charge inside the mitochondria. The oxonols are represented by a family of
 structures with the following general formulas (1 and 2):
 ##STR1##
 In these formulas, Q is either O or S and each R is independently chosen
 from an alkyl or aryl group of 1 to 20 carbon atoms. These compounds are
 commercially available through various sources including Molecular Probes,
 Inc. (Eugene, Oreg.).
 The bis-isoxazolone oxonols of Formula 1, namely Oxonol V and Oxonol VI
 (FIG. 2), have been used for measuring membrane potentials mainly by
 absorption rather than fluorescence [Salvador et al., J. Biol. Chem.
 273:18230-18234, 1998; Smith et al., J. Memb. Biol. 46:255-282, 1979]
 (incorporated herein by reference). The bis-barbituric acid oxonols of
 formula 2 are used for monitoring predominantly plasma membrane by changes
 in fluorescence intensity upon cell membrane depolarization or
 hyperpolarization [Epps et al., Chem. Phys. Lipids 69:137-150, 1994;
 Brauner et al., Biochim. Biophys. Acta. 771:208-216, 1984] (incorporated
 herein by reference).
 Another class of widely used membrane permeable dyes comprises carbocyanine
 derivatives of the following general Formula 3:
 ##STR2##
 In this formula, Q is O, S or C(CH.sub.3).sub.2 and each R is independently
 an alkyl group of 1 to 20 carbon atoms. These dyes, Indo- (DiI), thia-
 (DiS) and oxa- (DiO) carbocyanines with R ranging from one to seven carbon
 atoms, were the first potential sensitive probes developed [Sims et al.,
 Biochemistry 13:3315-3330, 1974] (incorporated herein by reference). These
 molecules, being positively charged, concentrate on the surface and inside
 the plasma membrane [Cabrini et al., J. Membr. Biol. 92:171-182, 1986]
 (incorporated herein by reference) and mitochondria [Bunting et al.,
 supra.] (incorporated herein by reference), where they aggregate with
 subsequent quenching of the fluorescence. This fluorescence intensity
 decrease is caused by potential-dependent binding of the molecules onto
 the membrane and aggregate formation [Guillet et al., supra.]
 (incorporated herein by reference). The main parameter used to monitor
 membrane potential in intact cells is the fluorescence intensity of dye in
 water, non-quenched phase of the dye. In this case it makes it impossible
 to distinguish between membrane potential changes of plasma membrane from
 that of mitochondria since an overall intensity for the cells is measured.
 Alternative approaches have been developed [U.S. Pat. No. 5,661,035;
 Gonzalez et al., Chem. Biol. 4:269-277, 1997; Gonzalez et al., Biophys. J.
 69:1272-1280, 1995] (incorporated herein by reference) that utilize
 Fluorescence Resonance Energy Transfer (FRET) phenomenon that can register
 membrane potentials in a ratiometric manner. These approaches and the
 matter compositions offered to practice FRET can elicit fast responses
 with high sensitivity to membrane potential changes. Unfortunately, these
 approaches and compositions are likely to cause artificial alterations in
 membrane structure and as a consequence, in cell functional behavior due
 to the unnatural incorporation of the highly hydrophobic dyes into the
 cell membrane. Additionally, use of fluorescently labeled lectin (WGA) as
 an affinity anchor for the second energy transfer counterpart reagent, can
 provoke cell functional responses by itself.
 SUMMARY OF THE INVENTION
 The present invention provides improved compounds and methods for measuring
 cell membrane electrical potential. In particular, the present invention
 uses donor and acceptor molecules to provide a fluorescent signal that is
 both rapid and sensitive to changes in membrane potential.
 One embodiment of the present invention is a method for identifying
 compounds having biological activity comprising combining living cells
 with a first membrane penetrative dye and with a second membrane
 penetrative dye to form a test cell mixture; combining the test cell
 mixture with a test compound to form a test cell/compound mixture; placing
 said test cell/compound mixture into a detection zone; and measuring a
 cellular response in said test cell. Preferably, the biological activity
 is an initiation of the cellular response. Advantageously, the biological
 activity is a block of the cellular response In one aspect of this
 preferred embodiment, the cellular response is measured by a change in
 plasma membrane electric potential of the cells. Preferably, the cells are
 in a suspension. Alternatively, the cells are adhered to a substrate. In
 another aspect of this preferred embodiment, the substrate is beads, a
 microscope slide or a well of a multi-well plate. Preferably, the test
 compound is in solution. In one aspect of this preferred embodiment, the
 test compound solution comprises a standard compound having known
 biological effect. Preferably, the standard compound is an ion channel
 opener, ion channel blocker, ion transporter blocker or ion pump blockers.
 Advantageously, the plasma membrane electric potential is measured by
 fluorescence energy transfer between the first membrane penetrative dye
 and the second membrane penetrative dye. Preferably, the first dye is a
 fluorescent lipophilic anion having a characteristic excitation maximum
 between about 300 nm and 800 nm. Advantageously, the second dye is a
 fluorescent lipophilic anionic molecule having a characteristic excitation
 maximum which overlaps with the emission spectrum of the first dye. In one
 aspect of this preferred embodiment, the second dye has a characteristic
 excitation maximum of between about 220 nm and 700 nm. Preferably, the
 test cell/compound mixture is contacted with light having a wavelength
 corresponding to the excitation spectrum of the first membrane penetrative
 dye, and the fluorescence intensity of the test cell/compound mixture is
 registered at a wavelength corresponding to the emission spectrum of the
 second membrane penetrative dye. Advantageously, the fluorescence
 intensity obtained on the test cell/compound mixture in the presence of
 the test compound is compared to the fluorescence intensity obtained on
 the test cell in the absence of the test compound. In one aspect of this
 preferred embodiment, the fluorescence intensity obtained on the test
 cell/compound mixture in the presence of a standard compound having a
 known cellular effect is compared to the fluorescence intensity obtained
 on the test cell in the absence of the standard compound. Preferably, the
 fluorescence intensity obtained from the test cell/compound mixture with
 the test compound and a standard compound having a known cellular effect
 is compared to the fluorescence intensity obtained on the test
 cell/compound mixture with the standard compound. Advantageously, the
 fluorescence emission intensity obtained on the test cell/compound mixture
 with the test compound is compared to the fluorescence emission intensity
 obtained on the test cell/compound mixture with the test compound and at
 least one standard compound having a known biological effect. A change in
 the fluorescence intensity indicates that the compound has an initiating
 effect on the cellular response, and a change in said fluorescence
 intensity indicates that the standard compound exerts the known biological
 response. The diminishing of the known effect induced by the standard
 compound in the presence of the test compound indicates that the test
 compound is an antagonist. The diminishing of the known effect induced by
 the test compound in the presence of the standard compound indicates that
 the test compound is an agonist.
 The present invention also provides a method for identifying compounds
 having biological activity comprising: combining living cells with a
 membrane penetrative dye to form a test cell mixture; combining the test
 cell mixture with a test compound to form a test cell/compound mixture;
 placing the test cell/compound mixture into a detection zone; and
 measuring a cellular response. Preferably, the biological activity is
 exerted through modification of plasma membrane electric potential of the
 cells. Advantageously, the biological activity is exerted through
 modification of mitochondrial membrane electric potential of the cells.
 Preferably, the cells are in a suspension. Alternatively, the cells are
 adhered to a substrate. The substrate is preferably beads, a microscope
 slide or a well of a multi-well plate. Advantageously, the test compound
 is in a solution. In one aspect of this preferred embodiment, the test
 compound solution comprises a standard compound having a known biological
 effect. Preferably, the standard compound is an ion channel opener, ion
 channel blocker, ion transporter blocker or ion pump blocker. Preferably,
 the test compound solution comprises a mixture of at least one test
 compound and at least one standard compound. In one aspect of this
 preferred embodiment, the cellular response is measured by change in
 electric potential across a membrane. Preferably, the membrane is the
 plasma membrane. Alternatively, the membrane is the mitochondrial
 membrane. In one aspect of this preferred embodiment, the membrane
 penetrative dye is a fluorescent lipophilic cation chosen from a group of
 fluorescent dyes whose spectral characteristics are different when in
 solution and when bound to the cell membranes. In one aspect of this
 preferred embodiment, the change in electric potential is measured by a
 change in fluorescence intensity of a membrane penetrative dye measured at
 least at two excitation and at least at two emission wavelengths.
 Preferably, at least one excitation and at least one emission wavelength
 is chosen from the set of wavelengths characteristic of aqueous form of
 the dye. In one aspect of this preferred embodiment, at least one
 excitation and at least one emission wavelength is chosen from the set of
 wavelengths characteristic of the membrane bound form of the dye.
 Advantageously, a change in ratio of fluorescence intensity measured at
 excitation and emission wavelengths characteristic of water soluble form
 of the dye to fluorescence intensity at excitation and emission
 wavelengths characteristic of the membrane bound form of the dye is
 indicative of both plasma membrane and mitochondrial membrane electric
 potential changes. A decrease in the ratio is indicative of plasma
 membrane depolarization, and an increase in the ratio is indicative of
 mitochondrial membrane depolarization.

DETAILED DESCRIPTION OF THE INVENTION
 The present invention provides methods for use in generating electric
 potential sensitive ratiometric changes in fluorescence of dyes in living
 cells or cell populations. In a preferred embodiment, the methods are used
 to identify compounds having biological activity, including initiation or
 blocking of cellular responses. In another preferred embodiment, the
 cellular response is measured by a change in plasma membrane electric
 potential of cells or cell lines. The cells and cell lines used with the
 method of the present invention may be prokaryotic or eukaryotic, and
 derived from plants, mammals and invertebrates, preferably humans. In one
 embodiment, the membrane potential sensor system comprises a first dye,
 which is preferably a membrane permeable fluorescent ion capable of
 redistributing between the cell cytoplasm and outer media upon changes in
 the membrane potential in accordance with its electrochemical potential,
 and a second dye, which is preferably a second penetrable fluorescent ion,
 preferably with the same charge sign as the first dye. The dyes are
 selected in such a way that one dye can be a donor and another reagent an
 acceptor of the light energy for fluorescence resonance energy transfer
 (FRET) to take place. When the cell has a resting potential of -60 mV
 across its plasma membrane (minus inside), the penetrable ion
 theoretically can have a 10-fold concentration gradient between external
 and internal water volumes. The same concentration gradient will exist
 across the cell membrane. When the membrane is depolarized, the penetrable
 ions redistribute evenly between the two membrane surfaces and the
 concentration of the fluorophore ions on the inside surface of the
 membrane increases. The potential sensitive fluorescent signal is created
 by fluorescence energy transfer between two neighboring molecules of the
 FRET pair. It is should be apparent to a person skilled in the art that
 the efficiency of the FRET is greatly dependent on the concentration of
 the interacting molecules and when the membrane is depolarized, the energy
 transfer between the donor and acceptor will increase as a product of the
 concentration of the dyes.
 More specifically, the present invention provides methods for use in
 generating electric potential sensitive ratiometric changes in
 fluorescence of dyes in single cells or cell populations. One method of
 the present invention comprises combining an energy acceptor dye and an
 energy donor dye with cells, irradiating the cells with light having a
 wavelength characteristic of the absorption spectra of the energy donor,
 and measuring fluorescence emission at two wavelengths characteristic of
 the emission spectra of the energy donor and the energy acceptor. The
 first dye is preferably a membrane penetrable fluorescent ion, preferably
 a lipophilic fluorescent ion, which redistributes between the outer and
 inner volumes of the cell upon changes in membrane potential in accordance
 with its electro-chemical potential. This first fluorescent dye is
 considered an energy acceptor. In a preferred embodiment, the first
 fluorescent dye has a characteristic excitation maximum between about 300
 and 800 nm. The second reagent, considered to be the energy donor, is also
 a membrane penetrable fluorescent ion, preferably lipophilic, and
 preferably with the same charge sign as the first fluorescent dye, to
 perform energy transfer to the first dye. In a preferred embodiment, the
 second fluorescent dye has a characteristic excitation maximum between
 about 220 and 700 nm. The schematic of the interaction between the probes
 in a membrane and generation of the electric potential sensitive signal is
 shown in FIG. 1. The resting potential of the cells has a negative charge
 localized inside; therefore, negatively charged probe molecules,
 represented here by members of the oxonol family of lipophilic fluorescent
 anions, are expelled from the cells and have a higher concentration in the
 media surrounding the cells compared to the intracellular concentration. A
 dye concentration gradient also exists across the membrane with a high
 concentration of the molecules in the outer leaflet and low concentration
 in the inner leaflet of the membrane. When the membrane is depolarized,
 the molecules located in the outer leaflet will move to the inner leaflet,
 and will be replaced from outside the cell with new molecules coming from
 the water space surrounding the cell. The total concentration of the
 molecules in the membrane will rapidly increase. Because of the increased
 concentration of both donor and acceptor molecules in the membrane, FRET
 occurs efficiently and produces a greatly enhanced signal in comparison to
 the signal associated with a polarized membrane.
 For measuring predominantly plasmalemma membrane potentials, the use of
 lipophilic anions is preferable to cations for several reasons. First, the
 highly negative potential in mitochondria prevents anions from entry and
 accumulation in the mitochondrial matrix. Second, lipophilic anions have
 better membrane penetrability than cations [Flewelling et al., Biophys. J.
 49:531-540, 1986] (incorporated herein by reference) and can concentrate
 better in the plasma membrane due to negative binding enthalpy compared to
 the positive (repulsive) binding enthalpy characteristic for cations
 [Flewelling et al., Biophys. J. 49:541-552, 1986] (incorporated herein by
 reference). Finally, they do not show significant disturbance of the
 membrane lipid bilayer [Bammel, Biochim. Biophys. Acta. 896:136-152, 1987]
 (incorporated herein by reference). It has also been shown that the
 lipophilic anions not only have stronger membrane binding but also have
 faster transmembrane migration rate compared with the positive hydrophobic
 cations [Franklin et al., Biophys J. 64:642-653, 1993] (incorporated
 herein by reference).
 In a preferred embodiment of the invention, both fluorescent ions are
 anions. When the cell membrane is polarized, it has a negative charge
 localized inside the cell and positive charge localized outside the cell.
 The intracellular negative charge prevents anionic reagents from entering
 into the cell, creating a concentration gradient of these reagents across
 the cell membrane. The concentration of the reagents is chosen in such a
 way that there is no energy transfer between them in the extracellular
 fluid. Upon depolarization, both reagents penetrate into the intracellular
 volume and their concentration in the cell membrane increases so that
 energy transfer between donor and acceptor molecules takes place. Because
 FRET is a bimolecular event, its efficiency will increase as a product of
 concentrations of the two dyes. Thus, if the concentrations of the two
 dyes are equal, then energy transfer efficacy would increase as the square
 of the dye concentration increase. In other words, two fold increases in
 dye concentration would cause a four fold increase in FRET efficiency.
 This type of non-linear dependency on the change in dye concentrations
 during membrane depolarization significantly augments the sensitivity of
 the method. The energy transfer that occurs between the two dye molecules,
 donor and acceptor, in the plasma membrane can be monitored by the change
 in the fluorescence intensity of the acceptor molecule while exciting the
 donor molecule with the wavelength that is not absorbed by the acceptor
 molecule.
 One skilled in the art will readily appreciate that the pair of
 donor-acceptor dyes for sensing membrane potential can be composed of pair
 of any negatively charged lipophilic molecules, with an energy donor and
 energy acceptor spectral characteristics fulfilling fluorescence energy
 transfer criteria. To fulfill this criteria the donor has to have an
 emission spectra overlapping with the absorption spectra of an acceptor
 molecule. The acceptor is also a negatively charged lipophilic anion with
 emission spectra shifted to the red compared to the donor emission
 spectra, so that its fluorescence intensity could be spectrally separated
 from the donor emission spectra and measured readily. The structures and
 excitation-emission spectra for a series of six exemplary oxonols,
 DiSBAC.sub.2 (3), DiSBAC.sub.4 (3), DiBAC.sub.4 (3), DiBAC.sub.4 (5),
 Oxonol V, and Oxonol VI, are presented in FIG. 2.
 Of course, oxonols suitable for use in the present invention as FRET
 sensors are not limited to the examples presented in FIG. 2. For this
 series of oxonols presented, or for any other fluorescent molecules, a
 person skilled in the art can appreciate that different combinations of
 potential sensor pairs can be used which have overlapping emission and
 excitation spectra. Such sensor pairs, whether constituting oxonol dyes or
 other compounds, are still considered to fall within the scope of the
 present invention. In a preferred embodiment, the emission spectrum of the
 donor and excitation spectrum of the acceptor overlap by about 50%, 40%,
 30% or 20%, more preferably by about 10% or 5%. Many fluorophores are
 known in the art or are commercially available from various sources.
 Donor-acceptor pairs suitable for use in the present invention can be
 readily determined by measuring excitation and emission spectra using a
 fluorescence spectrometer and determining whether there is overlap between
 the two.
 For simultaneous measurement of both plasma membrane and mitochondria
 membrane potentials, lipophilic cations are preferred. The cations, having
 a positive charge, are redistributed by concentrating in the cytoplasm and
 in mitochondria corresponding to their relative electric potentials. Thus,
 three concentrations of the probe are in a steady-state equilibrium with
 each other: the external concentration (water volume), the cytoplasmic
 concentration, and the intra-mitochondrial concentration. In accordance
 with the Nernst equation, each 60 mV of membrane potential brings about
 10-fold concentration difference of a permeable ion across the membrane.
 The plasma membrane potential of excitable cells is about 60 to 120 mV and
 the membrane potential of mitochondria is about 180 to 240 mV. This
 creates approximately 10 to 100 fold concentration gradient of the
 permeable cation across the plasma membrane and 1,000 to 10,000
 concentration gradients on the mitochondria membrane. It is well
 established that upon an increase in concentration, the carbocyanine dyes
 aggregate and their fluorescence is quenched. Upon depolarization, the dye
 is redistributed back into the extracellular space and its fluorescence
 intensity increases. Conventionally, the change in the fluorescence
 intensity of dye in the outer volume is measured to indicate the level of
 membrane polarization. These fluorescence intensity changes are identical
 for depolarization of both plasma membrane and mitochondria and cannot be
 used to differentiate depolarization events in both of these sites.
 In another preferred embodiment, the fluorescence intensity of lipophilic
 cations, preferably carbocyanine dyes, is measured at two different
 excitation/emission wavelength pairs, one characteristic of dye in
 solution and another of dye bound to the cell membrane and other
 intracellular structures. While the fluorescence intensity at "water"
 excitation/emission wavelengths allows monitoring changes in the membrane
 potential of both the plasmalemma and the mitochondria, the fluorescence
 intensity at "bound" excitation/emission wavelengths monitors
 predominantly mitochondrial membrane potential. By simultaneously
 registering the fluorescence intensity at both wavelength pairs, it is
 possible to distinguish membrane potential changes in both plasmalemma and
 mitochondria.
 In a preferred embodiment, living cells are first incubated with a membrane
 penetrative dye, or with a suitable combination of fluorescent
 donor-acceptor dye pairs to form a test cell mixture. A test compound with
 unknown effect is then added to the above mixture to form a test
 cell/compound mixture. The mixture is then placed into a detection zone in
 order to detect/measure a cellular response to the test compound. In a
 preferred embodiment, the test compound is in solution. The detection zone
 is where the sample is analyzed by the detection system which may be, for
 example, a cuvette of a spectrofluorometer, or an optical well in a
 96-well plate fluorescence reader. The cellular response, as reflected in
 a change of plasma membrane electric potential, is preferably measured by
 fluorescence energy transfer between the first membrane penetrative dye
 acting as a donor and the second membrane penetrative dye acting as an
 acceptor. The apparatus described in U.S. Pat. No. 5,804,436, the entire
 content of which is incorporated herein by reference, for example, can be
 used to conveniently measure the response. Cells grown in suspension or
 adhered to a substrate such as a well, preferably the bottom of a well
 (round or flat) of a multi-well plate may be used. Cells grown on a
 microscope slide or coated onto beads are also suitable for the analysis
 in accordance with the present invention. A variety of compounds with
 known effect on trans-membrane potential can be used as standard
 compounds, such as openers or blockers of ion channels, blockers of ion
 transporters or ion pumps.
 The modification of electrical potential of the plasma membrane can occur
 as a result of change in the activity of ion channels such as sodium,
 potassium, chloride and calcium channels. The change in the activity of
 voltage operated or chemo-regulated channels can also bring about change
 in electrical potential of plasma membrane. Ion channels, upon activation,
 allow for the ions to move across the cell membrane in accordance with
 their electrochemical potentials. There are two main types of ion
 channels: voltage operated and ligand-gated. Voltage operated channels are
 activated to the open state upon changes in transmembrane electric
 potential. Sodium channels in the neuronal axon or L-type calcium channels
 in neuromuscular junctions exemplify this kind of channel. Ligand-gated
 channels are activated to the open state upon binding a certain ligand
 with the chemoreceptor part of their molecules. The classical example of a
 ligand-gated channels is the nicotinic cholinergic receptor which, at the
 same time, is the sodium channel.
 Ion transporters represent another group of membrane transport assembly
 which can affect plasma membrane electrical potential in accordance with
 the present invention. Ion transporters use the electrochemical energy of
 transmembrane gradients of one ion species to maintain gradients of other
 ion counterpart. For example, the Na+/Ca.sup.2+ -exchanger uses the
 chemical potential of the sodium gradient directed inward to pump out
 calcium ions against their chemical potential. Other ion transporters
 include Na+/Cl.sup.-, Ca.sup.2+ /H.sup.+, HPO.sub.4.sup.2-, and the like.
 Ion pumps exemplify another category of membrane transport assembly that
 can modify electrical potential of plasma membrane in accordance with the
 present invention. Ion pumps act to maintain transmembrane ion gradients
 utilizing ATP as a source of energy. Ion pumps include, but are not
 limited to, Na+/K+-ATPase for maintaining transmembrane gradient of sodium
 and potassium ions, Ca.sup.2+ -ATPase for maintaining transmembrane
 gradient of calcium ions and H+-ATPase for maintaining transmembrane
 gradient of protons.
 In another preferred embodiment, trans-membrane electric potential is
 monitored by a method comprising the steps of: (a) combining a homogeneous
 suspension of living cells with membrane penetrative sensing dyes to form
 a cell/dye mixture, (b) combining the cell/dye mixture with a test
 compound having an unknown cellular effect to form a test mixture, (c)
 directing the test mixture through a detection zone; and (d) measuring a
 cellular response of the suspended cells to the test compound as the test
 mixture is flowing through the detection zone. The method will often
 include the additional steps of: (e) combining a homogeneous suspension of
 the cells with a standard compound having a known effect on the cellular
 response of the cells to form a standard mixture; (f) directing the
 standard mixture through the detection zone; and (g) measuring the
 cellular response of the cells to the standard compound. In one
 embodiment, the standard compound and the test compound are simultaneously
 mixed with the cells in the combining steps, and the measuring step
 detects the known effect or an alteration of the known effect. The
 standard compound can be an agonist or antagonist of the cellular
 response. In one mode of operation, steps (b), (c) and (d) are performed
 first, and then steps (e), (f), and (g) are performed using a single
 suspension of the cells prepared in step (a). In another mode of
 operation, steps (b) and (e) are performed simultaneously; steps (c) and
 (f) are performed simultaneously; and steps (d) and (g) are performed
 simultaneously. If the cellular response is detected in step (d) to
 indicate that the test compound is active to generate the response, and
 the standard compound is an antagonist, then a decrease in the cell
 response from step (d) of the first mode to combined steps (d) and (g) of
 the second mode, is indicative that the test compound is an agonist of the
 known effect. If the cellular response is not detected in step (d),
 indicating that the test compound is not active to generate the response,
 and the standard compound is an agonist, then an alteration of the known
 effect detected in step (g) of the first mode as detected in combined
 steps (d) and (g) of the second mode, is indicative that the test compound
 is an antagonist of the known effect. Preferably, the method is performed
 automatically under the direction of a programmable computer on a
 plurality of test compounds and a plurality of standard compounds, and a
 successive series of known antagonists is automatically added as the
 standard compound in step (e) if the cellular response is detected in step
 (d) to indicate that the test compound is active to generate the cellular
 response, whereby a decrease in the cellular response detected in combined
 steps (d) and (g) is indicative that the test compound is an agonist of
 the known effect; and a series of known agonists is automatically added as
 the standard compound in step (e) when the cellular response is not
 detected in step (d), whereby an alteration of the known effect detected
 in combined steps (d) and (g) is indicative that the test compound is an
 antagonist of the known effect
 EXAMPLE 1
 To characterize the spectral compatibility of the anionic lipophilic dyes
 for exploiting the FRET technique, the excitation and emission spectra
 were measured in the presence of PC 12 cells (American Type Culture
 Collection, No. CRL 1721). A suspension of cells in hybridoma medium
 (Gibco BRL) containing 1.5.times.10.sup.5 cells/ml was mixed with the
 respective dye (final concentration 0.5 .mu.M) for 30 min in a dark
 container. After the incubation, 3 ml of the suspension was transferred
 into a rectangular cuvette (1.times.1.times.4 cm) and the excitation and
 emission spectra were recorded using a FluoroMax-2 fluorometer
 (Instruments SA). The results are shown in FIG. 2.
 As can be readily appreciated by one skilled in the art, a variety of dye
 combinations with energy donor and acceptor compatibility can be construed
 from the dyes shown in FIG. 2, but not so limited. FIG. 3 presents
 excitation spectra for several donor/acceptor pairs registered either in
 PBS buffer or in PBS buffer in the presence of PC12 cells
 (2.5.times.10.sup.5 cells/ml). All dyes were used at a concentration of
 0.5 .mu.M. FIG. 3a shows excitation spectra registered at 600 nm for
 DiBAC.sub.4 (3) as the energy donor and DiSBAC.sub.2 (3) as the energy
 acceptor. FIGS. 3b and 3c show excitation spectra registered at 650 nm for
 DiBAC.sub.4 (3) and DiSBAC.sub.2 (3), respectively, as the energy donors
 and DiBAC.sub.4 (5) as the energy acceptor. FIGS. 3d and 3e show
 excitation spectra registered at 660 nm for DiBAC.sub.4 (3) and
 DiSBAC2(3), respectively, as the energy donors and Oxonol-V as the energy
 acceptor. For all the pairs presented, the emission wavelengths are chosen
 from the data presented in FIG. 2 in such a way that there is preferably
 no emission of the donor molecule.
 Those skilled in the art can readily appreciate that excitation spectra of
 the dyes in buffer show only one maximum characteristic for the excitation
 of the acceptor molecule. Upon cell addition, the intensity of the
 fluorescence increases and a second maximum appears, which is
 characteristic for the excitation of corresponding donor molecule. The
 appearance of the "donor" maximum at the emission wavelength where donor
 does not emit light, reflects the occurrence of the FRET when cells are
 present.
 EXAMPLE 2
 FIG. 4a represents excitation spectra measured at 650 nm, for the pair
 DiBAC.sub.4 (3)/DiBAC.sub.4 (5) (donor/acceptor) in the PC12 cell
 suspension in the presence and in the absence of 50 mM KCl, which is known
 to depolarize the cells. Cell membrane depolarization leads to additional
 increase of both fluorescence intensity of the acceptor molecule itself
 (peak at 600 nm) as well as FRET intensity (peak at 500 nm). After
 dividing the spectra of cells depolarized with KCl into the spectra of
 normal cells, represented in FIG. 4b, the increase in the FRET intensity
 was more pronounced (reaching a two-fold increase) than the increase in
 the fluorescence intensity of the dye itself. In another set of
 experiments, an uncoupler of oxidative phosphorylation, DNC, was added to
 the cells to depolarize mitochondria membrane. The spectral ratio (FIG.
 4b) did not show any increase in the fluorescence intensity or in the FRET
 at any wavelength. This experiment shows that with the anionic lipophilic
 dyes, the FRET technique can monitor changes exclusively in plasmalemma
 membrane potential and is not obscured by changes in the mitochondria
 membrane potential changes.
 EXAMPLE 3
 FIG. 5 illustrates K.sup.+ induced time dependent changes in the PC12 cell
 membrane potential which were registered using the donor/acceptor pair,
 DiBAC.sub.4 (3)/DiBAC.sub.4 (5). Cells were added to the solution of the
 two dyes and then a solution of KCl was added to provide a final dye
 concentration of 50 mM. Changes in fluorescence intensities of individual
 dyes (.lambda..sub.ex =490 nm and .lambda..sub.em =520 nm for DiBAC.sub.4
 (3) and .sub.ex =570 nm and .sub.em =650 nm for DiBAC.sub.4 (5)) as well
 as intensity of FRET (.lambda..sub.ex =490 nm and .lambda..sub.em =650 nm)
 were constantly monitored. Both dyes responded to the addition of cells
 and their subsequent depolarization with potassium chloride by increasing
 the corresponding fluorescence intensity. An important advantage of the
 present method is that the FRET-based signal intensity (490.sub.ex
 /650.sub.em) is significantly more sensitive in detecting the presence of
 cells and cell membrane depolarization than by detection of the
 intensities of individual dyes.
 EXAMPLE 4
 The HT-PS 100 system described in U.S. Pat. Nos. 5,804,436 and 5,919,646,
 the entire contents of which are incorporated herein by reference, was
 used to register concentration dependent changes in plasmalemma membrane
 potential upon gradual addition of adenosinetriphosphate, ATP, to the PC12
 cells. In the HT-PS 100 system, cells in the hybridoma media (600,000
 cells/ml), solution of dyes in PBS (0.5 .mu.M each) and solution of ATP in
 PBS (20 mM) are constantly mixed together in a flow. The components are
 mixed in a volumetric proportion of 1:1:1, the concentration of the ATP
 being changed exponentially during the registration time. The flow diagram
 of the system's fluidics is shown in FIG. 6. After the three reagent
 components are combined together, the resulting mixture flows through
 reaction chamber to allow development of the membrane potential changes to
 occur. With this set-up, the time between the cell activation with the ATP
 and registration of the membrane potential is always constant at any ATP
 concentration (60 seconds).
 FIG. 7 represents triplicate measurements of dose dependent ATP (A) and
 potassium (B) induced changes in the plasma membrane potential of the PC12
 cells registered by FRET using the DiBAC.sub.4 (3)/DiBAC.sub.4 (5) dye
 combination as the donor/acceptor pair. ATP causes saturation type of the
 membrane depolarization with its concentration, characteristic of binding
 to the limited number of receptor sites. In contrast, the potassium effect
 is non-saturable in nature, as is characteristic of the potassium
 diffusion potential. This approach is very sensitive and allows
 measurement of physiologically relevant changes in plasma membrane
 potentials with high precision, with the signal/noise ratio being equal to
 110 in these experiments.
 EXAMPLE 5
 The cationic lipophilic dye DISC.sub.3 (5) was used in this example. FIG. 8
 shows a series of excitation and emission spectra of the dye in a medium
 without cells and in the presence of PC12 cells (1.5.times.10.sup.5
 cells/ml). Cells were preincubated with the dye for 1 hour in a dark flask
 at different dye concentrations. After the incubation, aliquots of the
 cells (3 ml) were transferred in the fluorometer cuvette and the
 excitation and emission spectra were registered. For comparison, the dye
 spectra were registered in the same medium in the absence of cells. FIGS.
 8a-c represent real recorded fluorescence traces and FIGS. 8d-f represent
 the same spectra normalized to their respective maxima. The normalization
 procedure usually simplifies recognition of spectral shifts. The addition
 of cells to the dye solution brings about significant quenching of the dye
 fluorescence [Bunting, et al., Biophys J. 56:979-993, 1989; Guillet et
 al., J. Membr. Biol. 59:1-11, 1981] (incorporated herein by reference).
 However, at low dye concentrations (up to 60 nM), there is a clear red
 spectral shift in the observable excitation maximum and emission maximum.
 As shown in FIG. 8f, at high dye concentrations the spectral shift is
 miniscule if at all. These data show that there are at least three forms
 of the dye that exist in the cell suspension, free "water" dye, with high
 quantum yield, an aggregated form of the dye, which is not fluorescent,
 and membrane bound dye with red shifted spectra. At low dye concentration,
 the main portion of the positively charged dye is concentrated into
 mitochondria and aggregates there because of its high concentration and
 limited solubility. A smaller portion of the dye is absorbed onto cell
 membranes and only a minor portion of the dye is in solution in free form.
 Under these conditions, the fluorescence is attributed predominantly to
 the membrane bound form of the dye. At higher dye concentrations, the
 limit of the dye uptake [Bunting et al., supra.; Guillet et al., supra.]
 is responsible for an increase in the free soluble dye concentration and,
 consequently, in concealing fluorescence of the membrane bound form of the
 dye. From the spectral data presented in FIG. 8, it can easily be
 appreciated by those skilled in the art that by choosing appropriate pairs
 of excitation and emission wavelengths one can simultaneously monitor
 fluorescence of "water" and membrane bound fractions of the dye.
 In the data presented in FIG. 9, the following wavelength pairs (.sub.ex
 /.sub.em) were chosen: 645 nm/660 nm for "water" dye (WD) and 675 nm/690
 nm for membrane bound dye (MBD). After cells were added to the dye, the
 fluorescence of the WD (645.sub.ex /660.sub.em) diminished with time while
 the dye penetrated into and accumulated in the cell plasma and
 mitochondria. As it is clear from the trace, it is "water" dye
 fluorescence that was dramatically diminished upon the addition of the
 cells, with the membrane bound dye fluorescence staying practically at a
 constant level. In accordance with the Nernst equation, about 60 mV of
 plasma membrane potential assures a 10 fold increase in the free cytoplasm
 concentration of the dye relative to external media. A potential of about
 180 mV across the inner mitochondria membrane creates a thousand fold
 increase in the mitochondria compartment relative to the cytoplasm
 concentration. When the dye concentration in the mitochondria reaches its
 solubility limit, the dye tends to aggregate with fluorescence quenching.
 Free dye concentrations in the media, in the cytoplasm and in mitochondria
 equilibrate with each other along with the dye aggregates and with the
 membrane bound dye when the ratio (about 1:10:10,000 in
 media/cytoplasm/mitochondria) of the free dye reaches values driven by
 respective membrane potentials. Upon addition of KCl, which depolarizes
 plasma membranes due to dissipating the trans-membrane potassium gradient,
 the fluorescence of "water" dye increases and fluorescence of the membrane
 bound dye decreases or stays constant so that the ratio between MBD and WD
 fluorescence decreases upon plasma membrane depolarization. When DNC, an
 uncoupler of mitochondrial oxidative phosphorylation, was added,
 fluorescence of both MBD and WD increased with the kinetic responses of
 the MBD being significantly faster than that of the WD. In this case, the
 ratio between fluorescence of the membrane bound and the "water" fractions
 increased momentarily and then returned to values characteristic of the
 depolarized cells.
 Although the invention has been described in detail with reference to
 certain particular embodiments thereof, it will be understood that any
 variations and modifications apparent to those of skill in the art will
 still fall within the spirit and scope of the invention as provided by the
 following claims.