WO2006134319A1

WO2006134319A1 - Assay method

Info

Publication number: WO2006134319A1
Application number: PCT/GB2006/002082
Authority: WO
Inventors: Michael A. Cousin; Gareth J.O. Evans
Original assignee: University Court Of The University Of Edinburgh
Priority date: 2005-06-11
Filing date: 2006-06-07
Publication date: 2006-12-21
Also published as: GB0511893D0

Abstract

Described is a method for monitoring cellular processes, in particular a method in which at least two cellular processes are monitored using fluorescent indicators .

Description

Assay Method

Field of the Invention

The invention relates to assay methods for monitoring cellular processes . In particular it relates to a method of monitoring at least two cellular processes using fluorescent indicators.

Background to the Invention

High Content Screening (HCS) has very rapidly become a valuable addition to the methodologies available for drug discovery programmes in the pharmaceutical industry. Advances in computing and fluorescence imaging technology have allowed HCS to sample thousands of candidate drugs daily to quickly assess their suitability for use in mammals. The main advantage of HCS over previous methodologies is that the effect of candidate drugs on a number of

i different processes can now be simultaneously monitored in situ.

To date, a number of HCS methodologies have employed the use of multiple fluorescent markers to identify a number of different cellular parameters .

Three way HCS screens have been devised that measure intracellular calcium, cell death and mitochondrial function (Abraham et al, Large-Scale Investigation of Cytotoxicity Enabled by Automated Simultaneous Monitoring of Multiple Intracellular Processes in Individual Cells, http: //www.cellomics .com/content/menu/Downloads/) . However, such assays have required the changing of filter blocks between the measurement of the different parameters, thus prolonging the time period over which the measurements must be taken.

To address the problem of monitoring a number of parameters simultaneously, methods have been devised in which separate groups of cells preloaded with different fluorescent indicators are stimulated simultaneously with the emitted signals from each group of cells monitored generally simultaneously. However, although such a method enables a number of parameters to be assayed simultaneously using fluorescent indicators, different cells are being used to monitor each parameter.

The combined use of calcium indicators to monitor calcium concentration and the styryl dye FMl-43 to measure synaptic vesicle recycling in the same neuronal cells has been described before (Haller,T., et al, Proc. Natl. Acad. Sci. U. S. A., 95 (1998) 1579-1584; Shorte,S.L. et al, Cell Calcium, 18 (1995) 440-454.; Nunez,L. et al Endocrinology, 141 (2000) 2012-2017.; Burrone,J. and Lagnado , L . , J. Physiol., 505 (1997) 571-584.) However, the published assay has complications with bleedthrough of fluorescence signal from one to the other.

The present invention solves a number of problems associated with the prior art methods .

Summary of the Invention

The present inventors have surprisingly discovered that, when using fluorescent indicators which are excitable in the ultra-violet spectra and emit in the red spectra, for example Fura-2 or a derivative thereof, to measure a cellular parameter, such as ion concentration, the fluorescence emitted at wavelengths at which the fluorescence is less than 20% of the total emission of its emission spectrum, may be used to accurately measure the cellular parameter and that such measurements may be combined with measurements of another parameter using a fluorescent dye which is excited by green wavelengths of the visible spectrum and emit in red wavelengths without bleedthrough of the emission fluorescence signals. Accordingly, in a first aspect of the present invention, there is provided a method for the detection of at least a first parameter and a second parameter in a cell, said method comprising the steps :

(i) providing a cell loaded with a first fluorescent indicator and a second fluorescent indicator, wherein the first fluorescent indicator is excited by light of a first range of wavelengths, wherein the first range of wavelengths is in the ultraviolet range, and wherein the second fluorescent indicator is excited by light of a second range of wavelengths, wherein the second range of wavelengths is in the green wavelengths of the visible spectrum (ii) stimulating the cell using light of a wavelength of one of the first and second range of wavelengths and measuring the fluorescence emitted in a third range of wavelengths, and (iii) stimulating the cell using light of a wavelength of the other of the first and second range of wavelengths and measuring the fluorescence emitted in the third range of wavelengths, wherein the light emitted in response to the light of a wavelength of the ultra-violet range is indicative of the first parameter and the light emitted in response to the light of a wavelength of the green range is indicative of the second parameter.

The method of the invention allows bleedthrough problems to be minimised and thus removes the need to use changes of filter blocks to separate the emission signals from each of the indicators, greatly simplifying assays and enabling more rapid monitoring of a number of parameters .

Thus, in preferred embodiments of the invention, the assay is performed in the absence of addition or subtraction of an emission filter between step (ii) and step (iii) .

Any suitable fluorescent indicator which is excited in the ultra-violet range and which emits in the red range of the visible spectrum may be used as the first fluorescent indicator. Similarly any suitable fluorescent indicator which is excitable in the green range of wavelengths and which emits in the red range may be used as the second fluorescent indicator.

As described above, the invention enables bleedthrough problems to be minimised. Accordingly, in one embodiment, the fluorescence emitted by the first fluorescent indicator in response to light of the second range of wavelengths is less than 1%, such as less than 0.5%, of the fluorescence emitted by the first fluorescent indicator in response to light of the first range of wavelengths. Similarly, in another embodiment of the invention, the fluorescence emitted by the second fluorescent indicator in response to light of the first range of wavelengths is less than 1%, such as less than 0.5%, of the fluorescence emitted by the second fluorescent indicator in response to light of the second range of wavelengths . In preferred embodiments, neither of the first and second fluorescent indicators is excited by wavelengths of light used in step (ii) or step (iii) to excite the other of the first and second fluorescent indicators .

In a preferred embodiment, the first fluorescent indicator is excitable at wavelengths in the range 250-400nm. Preferably said indicator is not excitable at wavelengths outside of this range.

In a preferred embodiment, the second fluorescent indicator is excitable at wavelengths in the range 450-575nm. Preferably said indicator is not excitable at wavelengths outside of this range.

In one embodiment, the first and second indicators emit at wavelengths in the range 450-600 nm and 600- 850nm respectively. In the methods of the invention, emission from the first and second indicators is measured at wavelengths greater than 550nm, preferably greater than 575nm. This may be achieved by use of a longpass emission filter which blocks emission of lower wavelengths, for example less than 550nm or less than 575nm.

Thus, in a preferred embodiment, the detection steps of step (ii) and step (iii) are performed in the presence of an emission filter block which blocks detection of wavelengths of light in the range <575 nm. The choice of indicator will depend on the parameter being measured.

In one embodiment of the invention, the first fluorescent indicator is Fura-2 or a derivative therof . Derivatives of Fura-2 include any ion indicator, the structure of which is based on Fura- 2. These include the Na⁺ indicator SBFI and the K⁺ indicator PBFI. In a preferred embodiment, the first parameter is intracellular calcium and the first fluorescent indicator is fura-2.

The measurement of intracellular free sodium is important in assessing, for example, epilepsy. The measurement of intracellular free potassium is useful in assessing, for example, neurodegeneration and excitotoxicity.

In one embodiment of the invention, the second fluorescent indicator is FM4-64, which can be used to monitor vesicle fusion; BODIPY 558/568, which tracks lipid hydrolysis; Di-8-ANEPPS, which may be used for membrane potential measurement; Lysotracker red, which can be used to monitor acidic organelles; Rhodamine 123 or TMRM, which can each be used to measure mitochondrial membrane potential; Calcium crimson or Calcium orange, which can be used in dual ion measurements; or MitoSox red, which may be used for superoxide detection. In one preferred embodiment, the second parameter is synaptic vesicle recycling and the second fluorescent indicator is FM4-64.

Using the method of the invention, the time period over which the two parameters may be measured may be made very short. This has the advantage that the relationship between cellular events may be observed almost simultaneously. For example, when using Fura-2 as the first fluorescent indicator and FM4-64 as the second indicator to observe neuronal cells, the relationship between individual calcium transients and synaptic vesicle recycling may be observed.

In preferred embodiments of the invention, the time between steps (ii) and (iii) is less than 5 seconds, for example less than 3 seconds, such as less than 2 seconds, for example less than 1.5 seconds.

The method of the invention may be further modified to measure a third parameter using a third fluorescent indicator, for example a cell viability indicator .

The third fluorescent indicator should either (i) not be excited in a range of wavelengths used to excite the first and second fluorescent indicators (ii) not emit at wavelengths used to measure emission from the first and second fluorescent indicators, or both (i) and (ii) . In a preferred embodiment of the invention, the third fluorescent indicator is excited at a wavelength in the blue region of the visible spectrum, preferably in the range 400-500nm. Preferably, in response to stimulation, the third fluorescent indicator emits light at wavelengths of the green region of the visible spectrum.

In one preferred embodiment, the third fluorescent indicator is the cell viability indicator Sytox Green. To measure the emitted fluorescence from the Sytox Green, which is excited at wavelengths in the range 400nm to 500nm, and emits in the range 500-600 run, a bandpass emission filter of 510-550nm is preferably employed. Although this requires a change in filter from that used while measuring emission from the first and second fluorescent indicators, the time delay is not important as the indicator is measuring cell viability.

The assay of the invention may be used with any suitable cells. These may be neuronal cells, muscle cells or any cell in primary culture or immortalised cell line. In preferred embodiments of the invention, the cells are neuronal cells. In one embodiment, the neuronal cells are cerebellar granule neurones .

In one preferred embodiment of the invention, the first fluorescent indicator is Fura-2, the second fluorescent indicator is FM4-64 and a third fluorescent indicator, Sytox Green is used. Such an assay uses a combination of fluorescent dyes that report changes in intracellular calcium, vesicle fusion (and thus neurotransmitter release) and cell viability. These three parameters are vital reporters of nerve cell function and are typically altered during neuronal disorders.

The assays of the invention may be used to monitor the effect of a drug on one or all of the parameters being detected or monitored. Thus, in certain embodiments of the invention, the method includes the step of adding a drug to the cells. The effect of the drug on the parameters may be assessed by comparing the measured parameters in the absence and presence of the drug.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis unless the context demands otherwise.

The invention will now be described further in the following non-limiting examples with reference made to the accompanying drawings in which:

Figure 1 illustrates the excitation and emission spectra for Fura-2, Sytox Green and FM4-64 respectively;

Figure 2 illustrates a schematic protocol for carrying out an assay using Fura-2, Sytox Green and FM4-64; Figure 3 illustrates displays a field of cerebellar granule neurones loaded with three different dyes. These are (A) FM4-64 - which reports synaptic vesicle exocytosis, (B) fura-2 - which reports changes in intracellular calcium and (C) Sytox Green - which reports cell death. The panel in D shows all three indicators together as a three colour overlay image red = FM4-64, green = fura-2 and blue = Sytox green.

Figure 4 illustrates simultaneous monitoring of neuronal response in real time.

Figure 5A illustrates microscopical images of intracellular calcium responses using Fura-2 in an assay of the invention; the cell bodies shown are of 5-10μm size;

Figure 5B illustrates microscopical images of synaptic vesicle recycling and exocytosis visualised using FM4-64 in the same assay as Fig 5A; the cell bodies shown are of 5-10μm size;

Figure 5C illustrates microscopical images of dead cells visualised using Sytox Green in the same assay as Fig 5A; the cell bodies shown are of 5-10μm size;

Figure 6 illustrates the results of testing for bleedthrough . Figure 7 illustrates the effect of (i) ionomycin and (ii) sucrose and then KCl on cells loaded with FM4- 64 and fura-2.

Figure 8 illustrates two alternative protocols for studying the effect of a drug on intracellular Ca²⁺ and exocytosis using an assay of the invention.

Examples

Figure 2 illustrates schematically one embodiment of an assay of the invention. The assay uses a combination of fluorescent dyes that report changes in intracellular calcium, vesicle fusion (and thus neurotransmitter release) and cell viability. These three parameters are vital reporters of nerve cell function and are typically altered during neuronal disorders. The excitation and emission spectra for the indicators used are shown in Figure 1.

The assay itself is straightforward. Cerebellar granule neurones were loaded with FM4-64, which is a fluorescent membrane indicator that selectively labels recycling synaptic vesicles during stimulation. FM4-64 is excited by green wavelengths of light in the visible spectrum. After FM4-64 loading the cells were also loaded with fura-2-AM, which is a well characterised calcium indicator that is excited by ultraviolet light. A drug can be added at this stage. Cells were then stimulated at 340nm/380nm to excite the fura -2 and emission was monitored at greater than 575nm. The cells were then stimulated at 550nm to excite the FM4-64 and emission monitored at greater than 575nm. The excitation and monitoring cycles were repeated every 2 seconds (800 ms for the fura-2 ratio pair and 400 ms for the FM4-64) over 240 seconds. -

As no filter change is required between measurements, the intracellular calcium response and vesicle exocytosis can be monitored almost simultaneously. This is possible because the particular combination of filters and dyes that allow the monitoring of fura-2 and FM4-64 emission without any "bleedthough" from one to the other. The assay was completed by changing to a bandpass emission filter which allows emission of wavelengths of only 510-55Onm and adding a cell death indicator (Sytox Green) to determine whether any test compound has caused cell damage. Sytox Green can be used in conjunction with the other dyes because its narrow excitation (blue) and emission (green) peaks allow separation of its signal from the calcium and exocytosis wavelengths. The wavelengths used to excite the Fura-2 and FM4-64 indicators does not excite the Sytox Green indicator. On excitation of the Sytox Green indicator, although some excitation may occur of the FM4-64 indicator, any emission produced from the FM4-64 indicator will not be measured as no emission will be produced by FM4-64 within the wavelengths of the bandpass emission filter used to measure emission from the Sytox Green indicator. The results of the assay are shown in Figures 3 and 4. Figure 3 shows fluorescent images corresponding to the fluorescence resulting from FM4-64 - which reports synaptic vesicle exocytosis, (B) fura-2 - which reports changes in intracellular calcium and (C) Sytox Green - which reports cell death. The panel in D shows all three indicators together as a three colour overlay image red = FM4-64, green = fura-2 and blue = Sytox green.

Figure 4 illustrates simultaneous monitoring of neuronal response in real time. Bottom panel shows a bright field image of a granule cell neurite field. The panel above shows the same field loaded with Sytox Green (note the 2 dead cell bodies in the field of view) . Changes in intracellular free calcium and synaptic vesicle exocytosis can also be monitored in real time by stimulating the cells with elevated KCl. The top panels show these responses monitored in real time using the indicators fura-2 and FM4-64. The dotted line indicates addition of 50 mM KCl, the open circles indicate synaptic vesicle exocytosis and the closed circles indicate increases in intracellular free calcium. Small square panels below the graphs indicate the fluorescence signal from the cells at different times during the stimulus .

Further microsopical images and real-time results obtained using the assay are shown in Figures 5A, 5B, and 5C. Figure 6 illustrates the results of an experiment to demonstrates that bleedthrough is minimised using the method of the present invention. Panel A illustrates that when only FM4-64 was added (which is excited by 550nm light) , there was no bleedthrough to the fura-2 channel (measured at 380nm excitation) . Panel B illustrates that when fura-2 was added (seen at 380nm excitation) only trace fluorescence was seen in the red channel (550nm excitation) . This is not believed to be a consequence of bleedthrough but merely of autofluorescence of the cells . Indeed when the pictures were analysed in close-up, no bleedthrough was observed.

Figure 7 illustrates that the dyes report completely independent events within the cell . In Figure 7A the cells have been loaded with FM4-64 and fura-2 and stimulated with the calcium ionophore ionomycin in the absence of extracellular calcium. On addition of calcium, a huge increase in the fura- 2 signal and a drop in the FM4-64 signal was seen.

Figure 7B illustrates cells which were loaded with the same dyes but this time stimulated firstly with sucrose (which only should alter exocytosis and not calcium) and then KCl (which should alter both) . As shown in the figure, as expected, the sucrose stimulation alter exocytosis and not calcium and as expected, the KCl stimulation affected both exocytosis and calcium. In an alternative embodiment, as illustrated in Figure 8, all three fluorescent indicators are loaded into the cells prior to addition of the drug. The cells are then stimulated and fluorescence monitored over a period of 4 minutes .

The assay provides a very efficient and useful means of reporting changes in intracellular calcium, vesicle fusion (and thus neurotransmitter release) and cell viability in real time. These three parameters are vital reporters of nerve cell function and are typically altered during neuronal disorders .

All documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Claims

A method for the detection of at least a first parameter and a second parameter in a cell, said method comprising the steps: (i) providing a cell loaded with a first fluorescent indicator and a second fluorescent indicator, wherein the first fluorescent indicator is excited by light of a first range of wavelengths, wherein the first range of wavelengths is in the ultraviolet range, and wherein the second fluorescent indicator is excited by light of a second range of wavelengths, wherein the second range of wavelengths is in the green wavelengths of the visible spectrum (ii) stimulating the cell using light of a wavelength of one of the first and second range of wavelengths and measuring the fluorescence emitted in a third range of wavelengths , and

(iii) stimulating the cell using light of a wavelength of the other of the first and second range of wavelengths and measuring the fluorescence emitted in the third range of wavelengths, wherein the light emitted in response to the light of a wavelength of the ultra-violet range is indicative of the first parameter and the light in response to the light of a wavelength of the green range is indicative of the second parameter.

. The method according to claim 1, wherein the fluorescence emitted by the first fluorescent indicator in response to light of the second range of wavelengths is less than 1% of the fluorescence emitted by the first fluorescent indicator in response to light of the first range of wavelengths .

3. The method according to claim 1 or claim 2 , wherein the fluorescence emitted by the second fluorescent indicator in response to light of the first range of wavelengths is less than 1% of the fluorescence emitted by the second fluorescent indicator in response to light of the second range of wavelengths .

4. The method according to any one of the preceding claims wherein neither of the first and second fluorescent indicators are excited by wavelengths of light used in step (ii) or step (iii) to excite the other of the first and second fluorescent indicators.

5. The method according to any one of the preceding claims, wherein the first fluorescent indicator is excitable by wavelengths in the range 250-400 ran and the second fluorescent indicator is excitable by wavelengths in the range 450-575 nm.

6. The method according to any one of the preceding claims wherein the first fluorescent indicator is Fura-2 or a derivative thereof.

7. The method according to claim 6, wherein the first fluorescent indicator is selected from the group comprising Fura-2, SBFI, PBFI.

8. The method according to claim 7 , wherein the first parameter is intracellular calcium and the first fluorescent indicator is Fura-2.

9. The method according to any one of the preceding claims wherein the second fluorescent indicator is FM4-64, BODIPY 558/568, Di-8-ANEPPS, Lysotracker red, Rhodamine 123, TMRM, Calcium crimson, Calcium orange or MitoSox red.

10. The method according to claim 9 wherein the second paramenter is synaptic vesicle recycling and the second fluorescent indicator is FM4-64.

11. The method according to any one of the preceding claims wherein the assay is performed in the absence of addition or subtraction of a filter between step (ii) and step (iii) .

12. The method according to any one of the preceding claims wherein the detection steps of step (ii) and step (iii) are performed in the presence of an emission filter block which blocks detection of wavelengths of light in the range <575 run.

13. The method according to claim 12 , wherein the filter block blocks emission of light of <575nm.

14. The method according to any one of the preceding claims wherein the time between steps (ii) and (iii) is less than 3 seconds.

15. The method according to any one of the preceding claims, wherein the cells are further loaded or preloaded with a cell viability indicator.

16. The method according to claim 15, wherein the cell viability indicator is a third fluorescent indicator which is excited in a range of wavelengths in which neither of the first and second fluorescent indicators are excited.

17. The method according to claim 15 or claim 16, wherein the cell viability indicator is excited at wavelength in the blue region of the visible spectrum.

18. The method according to any one of claims 15 to 17, wherein, in response to stimulation, the cell viability indicator emits light at wavelengths of the green region of the visible spectrum.

19. The method according to any one of claims 15 to 18, wherein the cell viability indicator is Sytox Green.

20. The method according to any one of claims 1 to 19, wherein at least one of steps (ii) and (iii) are carried out in the presence of a candidate drug and the method is for monitoring the effect of the candidate drug on one or more of said first and second parameters .

21. The method according to claim 20, wherein steps (ii) to (iii) are performed in the absence of the candidate drug and then steps (ii) and (iii) are repeated in the presence of the candidate drug, wherein the difference in responses in the absence and presence of the candidate drug is indicative of the candidate drug effect.

22. The method according to claim 20, wherein steps (ii) to (iii) are performed in the presence of the candidate drug and then steps (ii) and (iii) are repeated in the absence of the candidate drug, wherein the difference in responses in the absence and presence of the candidate drug is indicative of the candidate drug effect.

23. The method according to any one of the preceding claims, wherein the cell is a neuronal cell .