Abstract:
The invention encompasses kits for an improved method for measuring membrane potential using compounds of the formula I as potentiometric probes. These probes may be used in combination with other fluorescent indicators such as Indo-1, Fura-2, and Fluo-3, such probes may be used in microplate reading devices such as FLIPR™, fluorescent imaging plate reader, sold by Molecular Devices Corp., of Sunnyvale, Calif.; flow cytometers; and fluorometers. Such probes are used to measure membrane potential in live cells 
     
       
                 
         
             
             
         
       
       
         
           
             wherein X is O or S; and 
             n is 1 or 2.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This is a divisional application of U.S. patent application Ser. No. 09/924,797, filed Aug. 8, 2001, now U.S. Pat. No. 6,852,504 to Klaubert et al., which is hereby incorporated by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Technical Field 
     The present invention relates generally to the fields of biology and chemistry, and bioanalytical instrumentation. In particular, the present invention is directed to composition and methods for use in sensing membrane potentials, especially in biological systems. Potentiometric optical probes enable researchers to perform membrane potential measurements in organelles and in cells that are too small to allow the use of microelectrodes. Moreover, in conjunction with imaging techniques, these probes can be employed to map variations in membrane potential across excitable cells and perfused organs with spatial resolution and sampling frequency that are difficult to achieve using microelectrodes. 
     2. Background of the Art 
     The plasma membrane of a cell typically has a transmembrane potential of approximately −70 mV (negative inside) as a consequence of K + , Na +  and Cl −  concentration gradients that are maintained by active transport processes. Potentiometric probes offer an indirect method of detecting the translocation of these ions. Increases and decreases in membrane potential (referred to as membrane hyperpolarization and depolarization, respectively) play a central role in many physiological processes, including nerve-impulse propagation, muscle contraction, cell signaling and ion-channel gating (references 1–3). Potentiometric probes are important tools for studying these processes, and for cell-viability assessment. Potentiometric probes include the cationic or zwitterionic styryl dyes, the cationic carbocyanines and rhodamines, the anionic oxonols and hybrid oxonols and merocyanine 540 (references 4–8). The class of dye determines factors such as accumulation in cells, response mechanism and toxicity. Mechanisms for optical sensing of membrane potential have traditionally been divided into two classes: sensitive but slow redistribution of permanent ions from extracellular medium into the cell, and fast but small perturbation of relatively impermeable dyes attached to one face of the plasma membrane (references 2 and 3). 
     The bis-barbituric acid and thiobarbituric oxonols, often referred to as DiBAC and DiSBAC dyes respectively, form a family of spectrally distinct potentiometric probes with excitation maxima covering most of the range of visible wavelengths. DiBAC 4 (3) and DiSBAC 2 (3) have been the two most popular oxonol dyes for membrane potential measurement (references 9 and 11). These dyes enter depolarized cells where they bind to intracellular proteins or membranes and exhibit enhanced fluorescence and red spectral shifts. Increased depolarization results in more influx of the anionic dye and thus an increase in fluorescence. DiBAC 4 (3) reportedly has the highest voltage sensitivity. The long-wavelength DiSBAC 2 (3) has frequently been used in combination with the UV light-excitable Ca 2+  indicators Indo-1 or Fura-2 for simultaneous measurements of membrane potential and Ca 2+  concentrations. Interactions between anionic oxonols and the cationic K + -valinomycin complex complicate the use of this ionophore to calibrate potentiometric responses. DiBAC and DiSBAC dyes are excluded from mitochondria because of their overall negative charge, making them superior to carbocyanines for measuring plasma membrane potentials. 
     In general, DiBAC and DiSBAC dyes bearing longer alkyl chains had been proposed to have better properties for measuring membrane potentials (references 5 and 12). DiSBAC 6 (3) has been selected to use in a FRET-based membrane potential assay (reference 12). There are no reports on DiBAC 1  and DiSBAC 1  for measuring membrane potential. 
     It has been discovered that DiBAC 1 (3) and DiSBAC 1 (3) that possess unexpected properties that can be used to measure membrane potentials with FLIPR and other fluorescence devices. Compared with other members of the DiBAC and DiSBAC family, DiBAC 1  (3) and DiSBAC 1 (3) give stronger signal and faster response, and exhibit greater water solubility. 
     SUMMARY 
     The invention encompasses an improved method for measuring membrane potential using compounds of formula I as potentiometric probes. These probes may be used in combination with other fluorescent indicators such as Indo-1, Fura-2, and Fluo-3, CALCIUM GREEN or Fluo-4. Such probes may be used in microplate reading devices such as the FLIPR fluorescent imaging plate reader, sold by Molecular Devices Corp. of Sunnyvale, Calif.; flow cytometers; and fluorometers. Such probes are used to measure membrane potential in live cells. 
     
       
                 
         
             
             
         
      
     
     The invention also encompasses test kits containing reagents of formula I, and reagents of formula I in combination with a another fluorescent reagent, in particular where the fluorescent reagent is a fluorescent indicator such as lndo-1, Fura-2, Fluo-3, CALCIUM GREEN or FIuo-4; (b) a second non-fluorescent reagent that can undergo energy transfer with the first compound; (c) a second nonfluorescent reagent that can absorb the excitation light or emission from the first compound; and/or (d) a second fluorescent or luminescent reagent that can undergo energy transfer with the first compound. 
     Another aspect of the invention involves a method for generating voltage-sensitive fluorescent changes comprising incubating the membrane with: 
     (a) A first reagent selected from the potentiometric probes which redistribute from one side of the membrane to the opposite side in response to transmembrane potential; and a second reagent selected from the group consisting of non-fluorescent dyes or pigments that are not membrane-permeable, and undergo energy transfer with the first reagent on one side of the membrane to reduce or eliminate the fluorescence signal on that side; or 
     (b) A first reagent selected from the potentiometric probes which redistribute from one side of the membrane to the opposite side in response to transmembrane potential; and a second reagent selected from the group consisting of non-fluorescent dyes or pigments that are not membrane-permeable, and that absorb the excitation light or emission from the first reagent on one side of the membrane to reduce or eliminate the undesired fluorescence signal; or 
     (c) A first reagent selected from the potentiometric probes which redistribute from one side of the membrane to the opposite side in response to transmembrane potential; and a second reagent selected from the group consisting of fluorescent or luminescent probes which undergo energy transfer with the first reagent, said second reagent being located adjacent to either the one side or the other side of the membrane. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  is a reaction scheme for making DiBAC and DiSBAC. 
         FIG. 2  illustrates the voltage-dependent fluorescence intensity changes of DiSBAC 1 (3) in P2X2 cells at different ATP concentrations. 
         FIG. 3  illustrates the absorption spectra of DiSBAC 1 (3), DiSBAC 2 (3), DiSBAC 4 (e), DiBAC 1 (3) and DiBAC 4 (3) in 1:1 methanol/water. 
         FIG. 4  illustrates the fluorescence spectra of DiSBAC 1 (3), DiSBAC 2 (3), DiSBAC 3 (3), DiBAC 4 (3) DiBAC 1 (3) and DiBAC 4 (3) in 1:1 methanol/water. 
     
    
    
     DETAILED DESCRIPTION 
     Compounds useful in practicing the present invention are made according to methods described by G. W. Fischer in Chem Ber, 1969, 102: 2609–2620, as shown in  FIG. 1 . 
     The invention is illustrated by the following examples: 
     EXAMPLE 1 
     Preparation of DiSBAC 1 (3) 
     
       
                 
         
             
             
         
      
     
     DiBAC and DiSBAC dyes are prepared based on the procedure for ethyl and butyl derivatives (H. Bartsch and G. Haubold, Arch. Pharm. 1982, 315, 761–766). Specifically, malonaldehyde bis(phenylimine)monohydrochloride (2.6 g, 10 mmol) and 1,3-dimethyl-2-thiobarbituric acid (3.5 g, 20 mmol) are dissolved in acetonitrile (40 mL). To the solution is added triethylamine (2 g, 20 mmol). The reaction mixture is refluxed until the starting materials are completely consumed as indicated by TLC. The mixture is cooled to room temperature, and poured into acidic water (pH 2–3, 350 mL). The resulting suspension is filtered to collect the solid that is washed with cold water and air-dried. The crude product is further purified on a silica gel column using a gradient of dichloromethanol/methanol to give the desired product. 
     DiSBAC 1 (5), DiBAC 1 (3), DiBAC 1 (5) and other oxonol dyes are prepared analogous to the above procedure. 
     EXAMPLE 2 
     Measuring Membrane Potentials using DiSBAC 1 (3) in Combination with the Fluorescence Imaging Plate Reader (FLIPR™) 
     This specific example illustrates how to use DiSBA 1 (3) in P2X2 cells in combination with the FLIPR™ fluorescent imaging plate reader sold by Molecular Devices Corp. of Sunnyvale, Calif. P2X2 cells are 1321 N1 astrocytoma cells transfected to overexpress the purinergic P2X2 ligand-gated ion channel. P2X2 belongs to a class of purinergic ion channels that pass calcium and sodium in response to purine, including adenosine 5′-triphosphate (ATP). P2X2 cells are propagated and maintained in DME (high glucose), 10% FCS, 1× Pen/Strep and 2 mM L-glutamine. Doubling time is approximately 36 hours. P2X2 cells should be split at a 1 to 2 ratio upon confluence. The cells should be carried for no more than 20 passages. When approaching this limit, a new frozen vial of cells should be resurrected. Following is a typical kit procedure:
         1. Plate 40,000 P2X2 cells in 100 μL per well for 96 well plates or 10,000 P2X2 cells in 25 μL per well for 384 well plates for overnight.       

     2. Prepare 1× Loading Buffer.
         2.1 To prepare the 1× Assay Buffer, pipette 10 mL of 10× Reagent Buffer (1× Hanks&#39; Balance Saline Solution+20 mM HPEPES, pH 7.40) and dilute in 90 mL of distilled water. Adjust pH to 7.4 using 1.0 N NaOH and/or 1.0 N HCl.   2.2 To prepare 10 mM stock solution of DiSBAC 1 (3), dissolve 3.8 mg DiSBAC 1 (3) in 1 mL DMSO   2.3 To prepare 10% pluronic acid, dissolve 400 mg pluronic acid in 4 mL water. Heat to 37° C. to complete dissolution.   2.4 To prepare 1× Loading Buffer, add 30 μL of stock DiSBAC 1 (3), 8 μL 10% pluronic acid, and 20.0 mg DB71 in the 1× FLIPR assay buffer.   3. Load Cells with 1× Loading Buffer   4. Remove cell plates from the incubator   5. Add 100 μL of 1× Loading Buffer per well of 96-well plates or 25 μL per well of 384-well plates.   6. Incubate plates at 37° C. for 30 minutes.   7. Run the FLIPR Membrane Potential Assay   7.1 Make 5× compound plate prior to running the FLIPR assay. Dissolve 27.5 mg of ATP (Sigma Cat# A3377) in 1 mL of sterile water to make a 50 mM stock solution. Make appropriate dilutions for 100 nM, 1 μM and 10 μM and transfer a minimum of 200 μL to each well of a compound plate.   7.2 Confirm that Membrane Potential Filter is installed. Choose p2x2.fcf for experimental setup in the FLIPR software. Set up the appropriate experiment parameters.   7.3 After incubation, transfer the plates directly to FLIPR and begin the Membrane Potential Assay.   8. Run FLIPR and perform data analysis.       

     The curve of the ATP dose response should look similar to that shown in  FIG. 2 ; an initial depolarization event depicted as an increase in fluorescence followed by repolarization or decay in signal near baseline. Also, EC50 should be approximately in the 10–100 nM range. 
     EXAMPLE 3 
     Measuring Membrane Potentials Using DiBAC 1 (3) in Combination with a Microscope 
     DiBAC 1 (3) is used to measure membrane potential change with a microscope as described by L. M. Loew (Methods in Cell Biology, vol. 38, pp 195–209). 
     EXAMPLE 4 
     Measuring Membrane Potentials Using DiSBAC 1 (3) in Combination with Flow Cytometer 
     DiBAC 1 (3) is used to measure membrane potential change with a flow cytometer as described by L. M. Loew (Methods in Cell Biology, Vol. 41, Part A, pp 195–209). 
     EXAMPLE 5 
     Water Solubility and Hydrophobicity Comparison of DiBAC and DiSBAC Dyes 
     DiSBAC 1 (3), DiSBAC 2 (3), DiSBAC 3 (3), DiSBAC 4 (3), DiBAC 1 (3) and DiBAC 4 (3) are dissolved in DMSO (3 mM). The DMSO stock solutions are respectively partitioned in 1:1 octanol/water mixture. The concentrations of the oxonol dyes in octanol and water layers are determined by absorption spectra. The results are summarized in the following table I. As shown in the table, DiSBAC 1 (3) and DiSBAC 1 (3) are much more hydrophilic than the other oxonol dyes. They also have much better water solubility. 
     
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                   
                   
                 Absorbance 
                   
               
               
                   
                 λ-max 
                 Absorb- 
                 Absorb- 
                 in Water/ 
                   
               
               
                   
                 octanol 
                 ance 
                 ance in 
                 Absorbance 
                 Relative 
               
               
                 Compound 
                 (nm): 
                 in Water: 
                 Octanol: 
                 in Octanol 
                 Values: 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 DiBAC 1 (3) 
                 495 
                 1.297 
                 0.3104 
                 4.179 
                 1 
               
               
                 DiBAC 4 (3) 
                 497 
                 0.002501 
                 2.854 
                 0.00088 
                 0.00021 
               
               
                 DiSBAC 1 (3) 
                 538 
                 0.06147 
                 0.9126 
                 0.0674 
                 1 
               
               
                 DiSBAC 2 (3) 
                 543 
                 0.01143 
                 2.187 
                 0.0052 
                 0.078 
               
               
                 DiSBAC 3 (3) 
                 544 
                 0.002256 
                 4.331 
                 0.0005 
                 0.0077 
               
               
                 DiSBAC 4 (3) 
                 544 
                 0.003318 
                 3.226 
                 &lt;0.0010 
                 &lt;0.0077 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 6 
     Absorption Comparison of DiBAC and DiSBAC Dyes 
     DiSBAC 1 (3), DiSBAC 2 (3), DiSBAC 3 (3), DiSBAC 4 (3), DiBAC 1 (3) and DiBAC 4 (3) are dissolved in methanol (1 mM). The stock solutions are diluted with 1:1 methanol/water, and the absorption spectra are recorded in a spectrophotometer. As shown in  FIG. 3 , DiBAC 1 (3) and DiSBAC 1 (3) possess unexpected blue shift compared to the other oxonol dyes. 
     EXAMPLE 7 
     Fluorescence Comparison of DiBAC and DiSBAC Dyes 
     DiSBAC 1 (3), DiSBAC 2 (3), DiSBAC 3 (3), DiSBAC 4 (3), DiBAC 1 (3) and DiBAC 4 (3) are dissolved in methanol (1 mM). The stock solutions are diluted with 1:1 methanol/water, and the absorption spectra are recorded in a fluorometer. As shown in  FIG. 4 , DiBAC 1 (3) and DiSBAC 1 (3) possess unexpected blue shift compared to the other oxonol dyes. 
     EXAMPLE 8 
     Fluorescence Response Comparison of DiBAC and DiSBAC dyes for Sensing Membrane Potentials in FLIPR Assays 
     DiSBAC 1 (3), DiSBAC 2 (3), DiSBAC 3 (3), DiSBAC 4 (3), DiBAC 1 (3) and DiBAC 4 (3) are dissolved in DMSO (1 mM). The stock solutions are respectively used to assay membrane potential changes in the P2X2 cells in combination with FLIPR™ as described in Example 2. The results are summarized in the following table 2. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                 Fluorescence enhancement by 10 
                   
               
               
                 Compounds 
                 μM ATP stimulation (in folds) 
                 Response speed 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 DiBAC 1 (3) 
                 3.7 
                 fast 
               
               
                 DiBAC 4 (3) 
                 3.3 
                 slow 
               
               
                 DiSBAC 1 (3) 
                 108.5 
                 fast 
               
               
                 DiSBAC 2 (3) 
                 38.6 
                 moderate 
               
               
                 DiSBAC 3 (3) 
                 14.5 
                 slow 
               
               
                 DiSBAC 4 (3) 
                 2.3 
                 slow 
               
               
                   
               
             
          
         
       
     
     As shown in the Table 2, DiSBAC 1 (3) is much more sensitive, and has faster response to membrane potential change than the rest of the DiSBACs. DiBAC 1 (3) also has much faster response to membrane potential change than DiBAC 4 (3). 
     EXAMPLE 9 
     Use of DiSBAC 1 (3) as a Fluorescent Indicator of Transmembrane Potential Depolarization of PC 12 Cells 
     Protocols for transmembrane potential measurements are summarized briefly since they are similar to those given in detail in Example 1 above. The Bis-(1,3-dimethylthiobarbituric acid)trimethine oxonol, (DiSBAC 1 (3)), fluorescent reagent may be purchased from Molecular Probes (Eugene, Oreg., USA). The 1× Cell-Loading Buffer for DiSBAC 2 (3), consists of sodium-free Tyrode&#39;s Buffer (SFTB), 2.5 μM DiSBAC 1 ,(3), and 200 μM Direct Blue 71 (as the fluorescence quencher). 
     A rat pheochromocytoma (adrenal) cloned cell line, PC12, is grown in RPMI 1640 culture medium with 10% fetal calf serum (FCS), 1% penicillin/streptomycin (PS), 2 mM L-glutamine, and 1 mM sodium pyruvate. Cells were grown in suspension, and subsequently centrifuged from growth medium and resuspended in DiSBAC 2 (3), 1× Cell-Loading Buffer. Approximately 100,000 cells were plated per well in a 96-well microtiter plate pre-coated with poly-D-lysine to enhance cell adhesion, centrifuged at 1000 rpm for 4 minutes, and placed in an incubator for an additional 20 minutes. Cells were not washed with any liquid medium, nor was the 1× Cell-Loading Buffer removed prior to performing fluorescence measurements. 
     The fluorescently labeled cells were analyzed for changes in membrane potential by using the FLIPR™ fluorescent imaging plate reader. Briefly, cells were depolarized with addition of 75 mM potassium gluconate in sodium-containing Tyrode&#39;s Buffer (SCTB). To inhibit voltage-gated sodium channels cells were previously incubated with 100 μM tetrodotoxin (TTX) for 5 minutes prior to depolarization. The data reveal that cell-depolarization (due to potassium addition) causes increased DiSBAC 1 (3) fluorescence. Inhibition of sodium channels by TTX results in smaller changes in membrane potential upon potassium addition as indicated by a smaller increase in fluorescence as compared to the positive control (75 mM potassium gluconate without TTX). 
     The above examples illustrate the present invention and are not intended to limit the invention in spirit or scope. 
     REFERENCES 
     
         
         
           
             1. Zochowski M, Wachowiak M, Falk C X, Cohen L B, Lam Y W, Antic S, Zecevic D., Imaging membrane potential with voltage-sensitive dyes.  Biol Bull  198, 1–21(2000). 
             2. Plasek J, Sigler K, Slow fluorescent indicators of membrane potential: a survey of different approaches to probe response analysis.  J Photochem Photobiol B  33, 101–124 (1996). 
             3. Loew L M., Characterization of Potentiometric Membrane Dyes. Adv Chem Ser 235, 151 (1994). 
             4. Wu J-Y, Cohen L B., Fast Multisite Optical Measurement of Membrane Potential. In Fluorescent and Luminescent Probes for Biological Activity, Mason W T, Ed., pp. 389–404 (1993). 
             5. Loew L M., Potentiometric Membrane Dyes. In  Fluorescent and Luminescent Probes for Biological Activity , Mason W T, 2nd Ed. Pp. 210–221 (1999). 
             6. Smith J C., Potential-sensitive molecular probes in membranes of bioenergetic relevance.  Biochim Biophys Acta  1016, 1–28 (1990). 
             7. Gross D, Loew L M., Fluorescent indicators of membrane potential: microspectrofluorometry and imaging. In  Methods Cell Biol  30, 193–218 (1989). 
             8. Freedman J C, Novak T S. Optical measurement of membrane potential in cells, organelles, and vesicles.  Methods Enzymol  172, 102–122 (1989). 
             9. Bronner C, Landry Y. The use of the potential-sensitive fluorescent probe bisoxonol in mast cells.  Biochim Biophys Acta  1070, 321–331 (1991). 
             10. Shapiro H M. Cell membrane potential analysis.  Methods Cell Biol  41, 121–133 (1994). 
             11. Loew L M. Confocal microscopy of potentiometric fluorescent dyes.  Methods Cell Biol  38, 195–209(1993). 
             12. Gonzalez J E, Tsien R Y. Improved indicators of cell membrane potential that use fluorescence resonance energy transfer.  Chem Biol  4, 269–277 (1997).