JPH1099096A - Number counting of survived cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply and rapidly counting the number of survived cells and/or the survival rate of cells. SOLUTION: This method for measuring the number of survived cells comprises measuring the strength of fluorescent light emitted from a measurement specimen treated with a nucleic acid fluorescent dyeing agent for dyeing only died cells and the strength of fluorescent light emitted from a measurement specimen subjected to a treatment for using the nucleic acid fluorescent dyeing agent and a treatment for injuring cell membranes, respectively, and subsequently comparing both the measured strengths with each other.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for easily and rapidly counting the number of surviving cells in a large number of samples by subjecting cells to a treatment with a nucleic acid fluorescent dye that stains only dead cells and a treatment to damage cell membranes. And / or a method for measuring cell viability.
It can be used for the evaluation of cytotoxicity in the fields of research and development of foods, medicine, pharmacy, biological research, and clinical examination and diagnosis.

[0002]

2. Description of the Related Art The safety and efficacy of pharmaceuticals, agricultural chemicals, cosmetics, foods, and the like are often evaluated by their cytotoxicity, and the number of viable cells is often measured as an indicator of this cytotoxicity. Methods for detecting and quantifying the number of viable cells or cell viability include trypan blue staining and MTT assay (Journal of Im).
Munological Methods, 65, 5
5-63, 1983), U.S. Pat.
No. 4,805, Molecular M
editine 32 pages 1340-1342, 19
Method described in 1995, U.S. Pat.
No. 16 has been proposed. Is a method of measuring the number of cells stained with the dye trypan blue that stains only dead cells blue within a visual field of a microscope, and the method is a method in which MTT is used for mitochondrial dehydrogenase in cells. The amount of blue-violet formazan that becomes a substrate and is generated as a result of the enzymatic reaction is quantified by an absorptiometer. In the method, two kinds of fluorescent substances (calcein AM and ethidium homodimer) are allowed to act on the same sample, This method measures the number of viable cells by simultaneously measuring the fluorescence emitted from living cells and the fluorescence emitted from dead cells. Calcein AM is a substance that rarely emits fluorescence, but is decomposed into calcein by the action of esterase in living cells, and emits strong fluorescence. The ethidium homodimer is a nucleic acid fluorescent stain having a property that it can pass only through a cell membrane having a damaged portion, specifically penetrates only dead cells and binds to nucleic acid, and emits fluorescence about 40 times as large as usual. These measurement methods have made it possible to easily and quickly measure the cytotoxicity of a drug or the like on a multiplate. In addition, the method described in
The intensity of the fluorescence emitted from the measurement sample subjected to propidium iodide staining only A, and the intensity of the fluorescence emitted from the measurement sample subjected to Hoechst 33258 staining DNA of dead cells and live cells were measured. This is a method of measuring cell viability by comparing both intensities. In addition, the method uses DNA from both live and dead cells.
U.S. Pat. No. 5,436,134 describes the intensity of fluorescence emitted by a measurement sample to which a cyanine-based nucleic acid fluorescent dye (Dye I) is applied and the DNA of dead cells only. No. 5,321,130, the intensity of the fluorescence emitted from a measurement sample that has been acted on by a cyanine nucleic acid fluorescent stain or a stain that emits fluorescence when applied to living cells (both are collectively called Dye II). And measuring the cell viability by comparing the two intensities.

[0003] However, in the above method, it is difficult to determine the staining with the dye trypan blue, and it is difficult to perform the measurement without the naked eye.
Since the methods of and basically depend on the enzymatic reaction,
It is easily affected by measurement temperature, measurement pH, enzyme reaction time, difference in enzyme activity depending on cell type, and the like. In addition, the method requires a centrifugal separation operation, and also requires an operation to dissolve poorly soluble formazan generated as a result of the enzyme reaction. In the method of (1), serum-derived esterase in the cell culture solution decomposes calcein AM, so that it is necessary to remove serum by washing the cells several times.However, since dead cells are easily removed by washing, Accurate measurement cannot be performed unless the system uses a cell culture solution of serum. (2) Measurement is complicated because two types of fluorescence are measured. Further, and in the methods,
(1) The operation is complicated because the measurement must be performed for two types of fluorescence. (2) When the cell viability is measured by applying two types of fluorescent dyes to one sample, the operation is performed later. There are drawbacks such as that the fluorescent dye that has acted before has an adverse effect on the fluorescent dye that has been made, and (3) it is necessary to prepare a calibration curve for each measurement in order to determine the viability.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have found that (1) the number of surviving cells in a sample of many specimens can be accurately measured simply and quickly, and (2) that serum is present in a cell culture medium. (3) Cell washing, cell centrifugation, dye lysis, and other complicated operations are not required for the measurement sample. (4) Measurement results are measured temperature, measurement pH, The present invention was completed as a result of various studies to provide a method for measuring the number of viable cells that satisfies such conditions as being less susceptible to differences in reagent reaction time and cell type.

[0005]

Means for Solving the Problems The present invention provides (1) the intensity of fluorescence emitted from a measurement sample to which a nucleic acid fluorescent dye that stains only dead cells has been applied, the treatment of the nucleic acid fluorescent dye, and the cell membrane A method for measuring the number of viable cells and / or cell viability, wherein the intensities of the fluorescence emitted by the measurement sample subjected to the treatment of damaging the cells are measured, and the two intensities are compared. The intensity of the fluorescence emitted by the measurement sample that has been subjected to the nucleic acid fluorescent stain to be stained and the intensity of the fluorescence that is emitted by the measurement sample that has been subjected to the stain and the agent that damages the cell membrane are measured, and the two intensities are compared. For measuring the number of living cells and / or cell viability by measuring the number of viable cells and / or cell viability including a nucleic acid fluorescent stain for staining only dead cells and an agent that damages cell membranes The kit and (3) (a) means for supplying a nucleic acid fluorescent stain for staining only dead cells to the sample to be measured, and (b) measuring the intensity of fluorescence emitted from the sample to be measured which has been acted on by the nucleic acid fluorescent stain Means, (c) means for damaging cell membranes, (d) intensity of fluorescence emitted by a sample to be measured which has been treated with a nucleic acid fluorescent dye which stains only dead cells and which has been treated to damage cell membranes. A number of viable cells and / or a means for comparing the intensities of the above (b) and (d) with respect to each of the measured fluorescence intensities. The present invention relates to an apparatus for measuring cell viability.

The measurement sample to which the present invention can be applied may be any cell having no cell wall, and examples thereof include animal cells, some microbial cells, and protoplasts of microorganisms and plants from which cell walls have been removed. . In particular, any animal cell can be used as a measurement sample, and examples thereof include animal body fluid cells (cells such as blood and lymph) and cancer cells. These samples can be anything that allows the cells to survive,
For example, cells in a culture solution and the like can be mentioned. When cytotoxic drugs, agricultural chemicals, cosmetics, foods, and the like coexist in the living environment of these samples, some or all of the cells in the samples become dead cells. If the number of viable cells is measured in such an environment, cytotoxicity can be evaluated.

Nucleic acid fluorescent dyes that stain only dead cells include: (1) whether they pass through a damaged cell membrane but do not pass through an intact cell membrane, or (2) do they emit weak fluorescence when not bound to nucleic acid? Or (3) emits strong fluorescence when bound to a nucleic acid. Specifically, ethidium homodimer,
Cationic nucleic acid fluorescent stains such as cyanine nucleic acid fluorescent stains; ethidium halides such as ethidium bromide;
A propidium halide such as propidium iodide can be used, but it is preferable to use ethidium homodimer or a cyanine nucleic acid fluorescent stain that stains only dead cells, and a cyanine nucleic acid fluorescent stain that stains only dead cells is preferred. Especially desirable. Examples of the cyanine-based nucleic acid fluorescent staining agent that stains only dead cells include those described in U.S. Pat. No. 5,321,130. Specifically, BOBO-1 Iodide and BOBO-3 I
oide, BO-PRO-1 Iodide, BO-
PRO-3 Iodide, POPO-1 Iodid
e, POPO-3 Iodide, PO-PRO-1
Iodide, PO-PRO-3 Iodide, TO
TO-1 Iodide, TOTO-3 Iodide,
TO-PRO-1 Iodide, TO-PRO-3
Iodide, YOYO-1 Iodide, YOYO
-3 Iodide, YO-PRO-1 Iodid
e, YO-PRO-3 Iodide (all trade names; manufactured by Molecular Probes) and the like. Fluorescence measurement wavelengths differ depending on the measurement conditions such as the type of cells to be measured and the type of target to be evaluated for cytotoxicity such as pharmaceuticals, agricultural chemicals, cosmetics, and foods, but each nucleic acid fluorescent stain has its own excitation. Since it has a wavelength and a fluorescence emission wavelength, it can be appropriately selected and used according to the measurement conditions. In addition, since the nucleic acid fluorescent dye itself may have an inherent fluorescence in addition to the object to be evaluated for cytotoxicity and the culture solution, the background fluorescence intensity (Fb) derived from these may be measured in advance using a fluorometer. Need to be kept.

[0008] The nucleic acid fluorescent dye has the property of passing through a damaged cell membrane, ie, the cell membrane of a dead cell, but cannot pass through an intact cell membrane, ie, a cell membrane of a living cell. When a stain is applied,
The stain selectively penetrates the dead cells, binds to the nucleic acid, and emits strong fluorescence. The number of dead cells can be quantified by measuring the intensity of this fluorescence (Fd ′) with a fluorimeter. Specifically, the fluorescence intensity (Fd) corresponding to the number of dead cells is obtained from the calculation formula of Fd = Fd'-Fb, and the number of dead cells can be quantified from this value using a calibration curve or the like. Further, the cell viability can be directly obtained without obtaining the number of dead cells.

[0009] As a method of damaging the cell membrane of the measurement sample, there are a method of applying an agent that damages the cell membrane, and a method of damaging the cell membrane by a physical method such as applying ultrasonic waves.

The agent that damages the cell membrane may be any agent as long as it damages the cell membrane of living cells and kills all living cells in the system. Surfactants; acids such as hydrochloric acid and sulfuric acid;
Bases such as sodium hydroxide and potassium hydroxide are exemplified. However, in order to clearly measure the fluorescence intensity described below, it is desirable to use a surfactant among the above-mentioned agents. Specifically, Triton X-100, Triton X-114, Triton X-305, Triton X -4
05, Brij-35, Brij-56, Brij-5
Polyoxyethylene ether type nonionic surfactants such as No. 8 (all trade names; manufactured by PIERCECHEMICAL); Tween-20, Tween-80, and Span-20 (all trade names; PIERCECHEMIC)
Ester type nonionic surfactant such as AL Co .; Nonidet p-40 (trade name; PIERCECHEMIC)
Alcohol type nonionic surfactants such as CHAPS, CHAPSO, BIGCHAP, DE
OXY-BIGCHAP (all brand names; PIERC
Cholic acid type nonionic surfactants such as ECHEMICAL); hexyl-β-D-glucopyranoside, octyl-β-D-glucopyranoside, octyl-β-glucoside, octyl-β-thioglupyranoside, octylglucopyranoside, etc. Glycoside nonionic surfactants; anionic surfactants such as N-lauroyl sarcosine, N-lauroyl sarcosine sodium, sodium lauryl sulfate, lithium lauryl sulfate, sodium dodecyl sulfate; saponins, panax ginseng saponins,
Examples thereof include saponins such as glycyrrhizin, glycyrrhizic acid and saikosaponins, and it is preferable to use a polyoxyethylene ether type nonionic surfactant or an alcohol type nonionic surfactant, and Triton X-100 (product) It is particularly desirable to use Triton X-114 (trade name) or Nonidet p-40 (trade name). These agents that damage cell membranes can be used alone or in combination.

When a drug that damages the cell membrane is acted on,
The drug can be made to act by dropping it as a solution of a predetermined concentration into a culture solution containing cells or the like that have been left standing, or dropping it into a stirred culture solution. Alternatively, a culture solution containing cells or the like may be gently added to a liquid containing the drug at a predetermined concentration, or the culture solution may be added and mixed with stirring. In such a case, the addition can be performed using, for example, a pipette, a syringe (syringe), a pipettor, a dispenser, a dropper, or the like. Alternatively, the cells may only need to be temporarily contacted with the drug that damages the cell membrane, eg, only immersed in the drug-containing solution. Preferably, it can be carried out by adding a predetermined amount of the drug solution to a culture solution according to a predetermined procedure using an automated device equipped with a microprocessor, and among devices known in the art, It can be selected and used appropriately.

When the cell membrane is damaged by a physical method, not only the cell membrane may be damaged but also DNA and the like in the cell may be destroyed. The processing conditions are examined as necessary. For example, when applying ultrasonic waves, it is possible to perform processing by immersing the vibrator in the sample or placing the sample in a cup-shaped container to which the vibrator is attached. A device capable of continuous processing can also be used, and can be used by appropriately selecting from devices known in the art. In order to further damage the cell membrane, an osmotic shock method, a freeze-thaw method, or the like can be used.

If the cell viability cannot be obtained directly from the measured values, a calibration curve is newly created for each processing condition as necessary, and the measured values are corrected.

When the cell membrane of a living cell present in the measurement sample after the measurement of the dead cell is damaged and the cell is killed, the fluorescent nucleic acid stain invades the former living cell, Binds to nucleic acids and emits fluorescence. Therefore, the measurement sample emits fluorescence that is enhanced by the fluorescence emitted by the living cells. By measuring the enhanced fluorescence intensity (Ft ′) with a fluorimeter,
The total number of cells present in the measurement sample can be quantified. Then, the number of living cells can be quantified by subtracting the number of dead cells from the total number of cells. Specifically, Ft = F
t′−Fb ′ and Fl = Ft−Fd = (Ft′−F
The fluorescence intensity (Ft) corresponding to the total number of cells and the fluorescence intensity (Fl) corresponding to the number of living cells were obtained by the calculation formula b ′) − (Fd′−Fb), and the total cell number and The number of viable cells, that is, the number of viable cells can be quantified by a calibration curve or the like. Fb 'is the background fluorescence intensity derived from the subject for which cytotoxicity is to be evaluated, the culture solution, the nucleic acid fluorescent dye, and the agent that damages the cell membrane. In many cases, when a surfactant is applied to damage the cell membrane, Fb becomes equal to Fb ′. In such a case, F1 can be obtained by a calculation formula of Fl = Ft′−Fd ′. .

[0015] Several measurement samples under the same conditions are prepared, and the number of dead cells is measured by the above-mentioned method for half of the samples, while the remaining half of the samples are treated with a nucleic acid fluorescent dye and the cell membrane is treated. And the total number of cells is determined in the same manner as in the above method. The number of viable cells can also be measured by subtracting the number of dead cells from the total number of cells thus determined. In this method, the step of measuring the number of dead cells and the step of measuring the total number of cells can be performed in parallel, so that efficient measurement can be performed. However, there is a possibility that problems such as the fact that measurement must be performed for many samples and the measurement error increases. On the other hand, when the total number of cells is measured after measuring the number of dead cells for the same sample, the time interval between the previous measurement step and the subsequent measurement step becomes a problem. The time interval varies depending on various conditions such as a cell type to be measured, a nucleic acid fluorescent dye, and a drug that damages a cell membrane, but is preferably short, preferably within 2 hours, and more preferably within 1 hour. Therefore, in consideration of various conditions such as a cell type to be measured, a nucleic acid fluorescent stain, and an agent that damages a cell membrane, it is desirable to select which method is the most appropriate measurement method for those conditions. .

The cell viability can be calculated from the quantified values of the total number of cells and the number of viable cells. In addition, the number of dead cells is almost 0.
When FlＦFt, it is assumed that Fl ≒ Ft. Therefore, when the fluorescence intensity is measured by simultaneously applying the nucleic acid fluorescent dye and the agent that damages the cell membrane at once, the total number of cells, that is, the number of viable cells can be measured only by one fluorescence measurement. The number can be quantified. Pharmaceuticals, pesticides,
In cosmetics, foods, and the like, cytotoxicity exerted by the number of viable cells and cell viability may be evaluated based on the values of the number of viable cells and cell viability. For example, the concentration IC _{50 of a} drug or the like in which 50% of cells are dead cells from the number of viable cells is determined. In some cases, according to the method of the present invention, their cytotoxicity can be evaluated simply and efficiently. In the present invention, the cell viability can be directly measured without calculating the number of viable cells.

The intensity of the fluorescence emitted from the measurement sample treated with a nucleic acid fluorescent dye that stains only dead cells;
A first step of measuring the intensity of the fluorescence emitted from the measurement sample subjected to the treatment with the nucleic acid fluorescent stain and the treatment to damage the cell membrane (this step includes each treatment performed before each measurement) In addition, the present invention can be carried out by a measuring instrument having a function of outputting the number of viable cells or the viability of cells by systemizing each operation in a series of steps including a second step for comparing both intensities. In the measuring device, a system in which a nucleic acid fluorescent dye for staining only dead cells and a drug for damaging cell membranes are set in advance to perform each treatment performed before each measurement in the first step of the system is used. It is desirable to practice the present invention. Since a kit for measuring the number of viable cells and / or a kit for measuring the cell viability can be applied to such a measuring device, the number of viable cells and / or the cell viability of the present invention can be measured efficiently. . Examples of the apparatus for automatically performing the measurement of the present invention include the apparatus shown in FIG. 1 and the apparatus shown in FIG. Although the device is referred to as a viable cell count measuring device, it may be understood as a cell viability measuring device or a viable cell count and / or cell viability measuring device. In the present description and in the claims, the device is to be understood in a broad sense.

FIG. 1 shows an example of an apparatus for measuring the number of viable cells in the case where the present invention is carried out by a method of measuring the number of dead cells for the same sample and then measuring the total number of cells. Thus, the device consists of a spectrofluorometer built into the device, where a measuring cuvette with a stirrer (6), an excitation-side spectrometer (14), a fluorescence spectrometer (15), Light source (10), lens (5, 9 and 1)
1), polarizing plates (4 and 12) and photomultiplier tube (1
3). In the figure, 1 indicates a drug that damages the cell membrane, and 3 indicates a nucleic acid fluorescent dye that stains only dead cells. Each drug is set in a tank or a detachable container in the apparatus. The medicine set in these tanks or detachable containers is supplied to a medicine injector (2).
Can be injected into the measuring cuvette (6) with a stirrer. The apparatus is provided with a valve and a pump (not shown) for appropriate control. Also,
Washing pump, drainage pump, switching valve,
An agitating air pump, a nozzle and the like may be provided. Further, reference numeral 7 in the figure denotes an apparatus for converting an analog signal into a digital signal, and reference numeral 8 denotes a control system. Further, the measurement cuvette (6) can be continuously moved as required, and can automatically measure a plurality of cuvettes. The measurement cuvette (6) can be adapted to be held in a thermostat. Furthermore, the measurement can be performed automatically by fixing the measurement cuvette (6) and allowing the drug injector and the spectrofluorometer to move continuously as required.

In FIG. 2, several measurement samples under the same conditions are prepared, and the number of dead cells is measured for the half of the samples by the above-mentioned method. The present invention is applied to a method of performing a treatment for acting and a treatment for damaging the cell membrane, measuring the number of dead cells for a sample in the case of obtaining the number of dead cells in the same manner as in the above method, and then measuring the total number of cells. An example of a viable cell number measuring device in the case of performing is shown. Thus, the device consists of a spectrofluorometer built into the device, where a 96-well microplate with plate agitator (1
6), excitation side spectrometer (14), fluorescence spectrometer (15), light source (10), lenses (5, 9 and 11), polarizing plate (4)
And 12) and a photomultiplier tube (13). Reference numeral 21 in the figure denotes a cell dilution suspension, which is set in a tank or a detachable container in the apparatus. A stirrer is installed in the tank or the detachable container to supply a uniform concentration suspension to each well. Reference numeral 1 denotes an agent that damages the cell membrane, and reference numeral 3 denotes a nucleic acid fluorescent dye that stains only dead cells. Each of the agents is set in a tank or a detachable container in the apparatus. The suspension or drug set in these tanks or detachable containers is dispensed by a dispenser (22) into a 96-well microplate with plate agitator (16).
Dispense into each well in. Dispenser (22)
A disposable tip or nozzle is attached to the tip of the, and these can be replaced or washed as necessary. Further, the tip portion of the dispenser (22) may be branched, and the medicine can be dispensed to a plurality of sample wells at the same time. In the figure, 7 is a device for converting an analog signal into a digital signal, and 8 is a control system. The microplate (16) can be moved continuously as required, and can automatically measure a plurality of microplates. Further, the microplate was fixed, and a dispenser (2
2) and the spectrofluorimeter can be moved continuously as needed so that measurements can be made automatically.

When the present invention is carried out by measuring the number of dead cells for the same sample and then measuring the total number of cells, it is efficient to carry out the measurement using the apparatus shown in FIG. is there. An example of how to use this device is shown below. [1] A measurement sample such as cells in a culture solution is set in a measurement cuvette (6) in which a stirrer is set, and stirred. Also, a fluorescent nucleic acid stain for staining only dead cells (3)
The drug (1) that damages the cell membrane is set in a tank in the apparatus in advance. [2] A nucleic acid fluorescent stain (3) for staining only dead cells is injected into a measurement cuvette (6) in which a measurement sample is set by a drug injector (2), and the measurement sample is stained. After that, the intensity of the fluorescence emitted by this is measured by a spectrofluorometer. The measurement result is converted from an analog signal to a digital signal and sent to the control system (8). The amount of the nucleic acid fluorescent dye (3) that stains only dead cells is automatically controlled in conjunction with the control system (8) for measuring the fluorescence intensity. [3] After the measurement in [2], the drug (1) that damages the cell membrane is injected into the measurement cuvette (6) by the drug injector (2), and all cells in the measurement sample are determined to be dead cells. You. After that, the intensity of the fluorescence emitted by this is measured by a spectrofluorometer. The measurement result is converted from an analog signal to a digital signal and sent to the control system (8),
The cell viability is measured in comparison with the measurement result of [2]. Further, if necessary, the cell number is known and the above measurement is performed on some samples having an appropriate cell number,
By giving a calibration curve to this apparatus, the number of viable cells and the like can be measured for a sample whose number of cells is unknown.

Several samples under the same conditions are prepared, and the number of dead cells is measured for the half of the samples by the above method, while the remaining half of the samples are treated with a nucleic acid fluorescent dye and the cell membrane is treated. When the total cell count is obtained in the same manner as in the above method, it is efficient to perform the measurement using the apparatus shown in FIG. An example of how to use this device is shown below. [1] In order to prepare a measurement sample under the same conditions, the measurement sample is used as a cell-diluted suspension (21) by a dispenser (22).
96-well microplate with plate agitator (1
Dispense into each well of 6). After dispensing, the microplate is stirred with a plate stirrer. The fluorescent nucleic acid stain (3) for staining only dead cells and the agent (1) for damaging cell membranes are set in a tank in the apparatus in advance. [2] A nucleic acid fluorescent stain (3) for staining only cells is dispensed by a dispenser (22) into a 96-well microplate (16) in which a measurement sample is set, and the measurement sample is stained. Then, the intensity of the fluorescence emitted from the sample in each well is measured with a spectrofluorometer. The measurement result is converted from an analog signal to a digital signal and sent to the control system (8). The amount of the nucleic acid fluorescent dye (3) used to stain only dead cells is determined by the control system for fluorescence intensity measurement (8).
Automatically controlled in conjunction with. The dispenser is provided with a separate dispensing tube for each drug, and the drug can be dispensed to a plurality of wells by one operation. [3] A sample to be measured under the same conditions as those used in the measurement of [2] is set by a dispenser with the agent (1) that damages the cell membrane and the nucleic acid fluorescent dye (3) that stains only dead cells. Into a 96-well microplate (16), and stain all cells in the measurement sample as dead cells. Then, the intensity of the fluorescence emitted from the sample in each well is measured with a spectrofluorometer. The measurement result is converted from an analog signal to a digital signal and sent to the control system (8). Drugs that damage cell membranes (1)
The amount of the nucleic acid fluorescent dye (3) used to stain only dead cells is automatically controlled in conjunction with the control system (8) for measuring the fluorescence intensity. The measurement result is converted from an analog signal to a digital signal and sent to the control system (8),
The cell viability is measured in comparison with the measurement result of [2]. Further, if necessary, the cell number is known and the above measurement is performed on some samples having an appropriate cell number,
By giving a calibration curve to this apparatus, the number of viable cells and the like can be measured for a sample whose number of cells is unknown.

As described above, according to the present invention, (a) means for supplying a nucleic acid fluorescent stain for staining only dead cells to a sample to be measured, and (b) a sample to be measured on which the nucleic acid fluorescent stain has acted Means for measuring the intensity of the emitted fluorescence, (c) means for damaging the cell membrane, (d) an object to be treated which has been treated with a nucleic acid fluorescent dye which stains only dead cells and which damages the cell membrane At least means for measuring the intensity of the fluorescence emitted from the sample is provided, and means for comparing both the intensity of the above (b) and the intensity of the above (d) for each measured intensity of the fluorescence is provided. A device for measuring the number of viable cells and / or cell viability is provided, each component of which is known in the fields of automated immunoassays, automated biochemical assays and the like. Thing It can be used by applying to choose the inner shell, or may be used in addition to appropriately modified as required. A means for supplying a nucleic acid fluorescent stain for staining only dead cells to a sample to be measured, preferably by a programmed computer, for example, a microcomputer, and (b) a sample to be measured with the nucleic acid fluorescent stain Means for measuring the intensity of the fluorescence emitted by (a), (c) means for enabling damage to the cell membrane, and (d) measurement which has been subjected to a treatment in which a nucleic acid fluorescent dye that stains only dead cells is caused to act and damage the cell membrane. A means for measuring the intensity of the fluorescence emitted from the target sample, and / or a plurality or all of the means for comparing the intensities of the above (b) and (d) for each of the measured intensities of the fluorescence Is controlled.

In the method for measuring the number of living cells and / or cell viability and the kit for measuring the number of viable cells and / or cell viability according to the present invention, the nucleic acid fluorescent dye for staining only the various dead cells described above is used. And the various agents that damage the cell membrane described above can be used in combination with each other, but the nucleic acid fluorescent stain that stains only the various dead cells described above and the various surfactants described above are used in combination. It is desirable to use. In addition, a mixture in which the nucleic acid fluorescent dye for staining only dead cells and the various surfactants described above are mixed in advance as necessary can also be used.

Further, the apparatus for measuring the number of viable cells and / or cell viability corresponding to the apparatus for measuring the number of viable cells (or apparatus for measuring the number of viable cells and / or cell viability) shown in FIG. 1 or FIG. One of the desirable forms of the kit includes a kit in which a nucleic acid fluorescent stain for staining only dead cells and an agent for damaging cell membranes are each pre-filled in a detachable container of the viable cell counting device. . In this case, the staining agent and the drug are filled in the respective detachable containers, but may be filled in the same detachable container as needed.

[0025]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Some embodiments of the present invention will be described below. [1]. The intensity of the fluorescence emitted by the measurement sample that has been subjected to the nucleic acid fluorescent stain that stains only dead cells, and the intensity of the fluorescence that is emitted by the measurement sample that has been subjected to the treatment with the nucleic acid fluorescent stain and the treatment of damaging the cell membrane A method for measuring the number of viable cells and / or the cell viability, wherein each is measured and the two intensities are compared. [2]. (i) a nucleic acid fluorescent dye that stains only dead cells is allowed to act on the measurement sample, and the intensity of the resulting fluorescence is measured. (ii) Then, a treatment for damaging the cell membrane of the sample is performed, The measurement method according to the above [1], wherein the intensity of the fluorescence enhanced by killing is measured. [3]. (i) treating a measurement sample with a nucleic acid fluorescent dye that stains only dead cells, measuring the intensity of the fluorescence emitted from the sample, and (ii) treating the other sample with the nucleic acid fluorescent dye. And treatment to damage the cell membrane,
The measurement method according to the above [1], wherein the intensity of the fluorescence emitted from the sample is measured. [4]. The method according to any one of the above [1] to [3], wherein the nucleic acid fluorescent stain is a cationic nucleic acid fluorescent stain. [5]. The measurement method according to any one of the above [1] to [4], wherein the cell membrane is damaged using an agent that damages the cell membrane. [6]. The method according to the above [5], wherein the agent that damages the cell membrane is a surfactant. [7]. The intensity of the fluorescence emitted by the measurement sample that has been subjected to the nucleic acid fluorescent stain that stains only dead cells, and the intensity of the fluorescence that is emitted by the measurement sample that has been subjected to the treatment with the nucleic acid fluorescent stain and the treatment of damaging the cell membrane The method according to any one of the above [1] to [6], wherein each operation in a series of steps comprising measuring each and comparing the two intensities is performed by an automated device. [8]. The intensity of the fluorescence emitted by the measurement sample subjected to the nucleic acid fluorescent stain that stains only dead cells and the intensity of the fluorescence emitted by the measurement sample subjected to the stain and the agent that damages the cell membrane were measured. Viable cell number and / or cell viability composed of a nucleic acid fluorescent stain that stains only dead cells and an agent that damages the cell membrane for measuring viable cell number and / or cell viability by comparing intensity Kit for rate measurement.

[0026]

[9]. The method according to any one of the above [1] to [3], wherein the nucleic acid fluorescent stain is a ethidium homodimer or a cyanine nucleic acid fluorescent stain that stains only dead cells. [10]. The above-mentioned [1] to [3], wherein the nucleic acid fluorescent stain is a cyanine nucleic acid fluorescent stain that stains only dead cells.
The measurement method according to any one of the above. [11]. The above-mentioned [5], [6], wherein the agent that damages the cell membrane is a polyoxyethylene-type nonionic surfactant or an alcohol-type nonionic surfactant.

The measuring method according to any one of [9] and [10]. [12]. The agent that damages the cell membrane is Triton X-10
0 (trade name), Triton X-114 (trade name) or Nonidet p-40 (trade name)
Above-mentioned [5], [6],

The measuring method according to any one of [9] and [10]. [13]. The kit according to the above [8], wherein the nucleic acid fluorescent stain is a cationic nucleic acid fluorescent stain. [14]. The kit for measuring the number of viable cells and / or cell viability according to the above [8], wherein the fluorescent nucleic acid stain is ethidium homodimer or a cyanine nucleic acid fluorescent stain that stains only dead cells. [15]. The kit for measuring the number of living cells and / or cell viability according to the above [8], wherein the nucleic acid fluorescent stain is a cyanine nucleic acid fluorescent stain that stains only dead cells. [16]. The survival according to any one of the above [8], [13], [14] and [15], wherein the agent that damages the cell membrane is a polyoxyethylene type nonionic surfactant or an alcohol type nonionic surfactant. A kit for measuring cell number and / or cell viability. [17]. The agent that damages the cell membrane is Triton X-10
0 (trade name), Triton X-114 (trade name) or Nonidet p-40 (trade name)
[8], [13], [14]
And the kit for measuring the number of living cells and / or the cell viability according to any one of [15] and [15]. [18]. (A) means for supplying a nucleic acid fluorescent stain for staining only dead cells to a sample to be measured, (b) means for measuring the intensity of fluorescence emitted from the sample to be measured with the nucleic acid fluorescent stain, and (c) Means for allowing cell membrane damage; (d) means for measuring the intensity of fluorescence emitted by a sample to be measured which has been treated with a nucleic acid fluorescent dye that stains only dead cells and has been treated to damage the cell membrane; A method for measuring the number of viable cells and / or cell viability, comprising at least a means for comparing the intensities of the above (b) and (d) with respect to each measured fluorescence intensity. apparatus. [19]. (i) a nucleic acid fluorescent dye that stains only dead cells is allowed to act on the measurement sample, and the intensity of the resulting fluorescence is measured. (ii) Then, a treatment for damaging the cell membrane of the sample is performed, [18] The measuring device according to the above [18], wherein the intensity of the fluorescence enhanced by killing is measured. [20]. The measuring device according to the above [18] or [19], wherein (i) and (ii) are performed in an automated form. [21]. [20] The above-mentioned [20], wherein each process is automatically controlled according to a program stored in advance using a microprocessor.
The measuring device as described. [22]. (A) a device for holding a drug that damages the cell membrane, (b) a device for holding a nucleic acid fluorescent dye that stains only dead cells, and (c) a sample to be measured with the drug from the device (a) or (b). [18] to [2], comprising: a drug injector to be supplied to the container, (d) a container such as a measurement cuvette or a microplate with a stirrer capable of holding a sample to be measured, and (e) a spectrofluorometer.
1] The measuring device according to any one of [1]. [23]. (A) For the same sample to be measured, (i)
After allowing a nucleic acid fluorescent stain that stains only dead cells to act and measuring the intensity of the resulting fluorescence, (ii)
(B) (i) a device that allows the cell membrane of this sample to be subjected to a treatment for damaging the cell membrane and killing the cells to measure the intensity of the enhanced fluorescence; A nucleic acid fluorescent dye that stains only dead cells is acted on, and the intensity of the resulting fluorescence is measured. (Ii) A nucleic acid fluorescent stain that stains only the dead cells is applied to another sample to be measured. [18] The measuring device according to [18], further comprising a device capable of performing a process of causing the cell membrane to be damaged and measuring the intensity of the fluorescence emitted as a result.

[24]. The substance required for toxicity evaluation is allowed to act on cells, and then the obtained measurement sample is caused to act on a nucleic acid fluorescent dye that stains only dead cells, and the intensity of the fluorescence emitted from the measurement sample and the nucleic acid fluorescent stain are measured. Measuring the intensity of the fluorescence emitted from the measurement sample subjected to the treatment for acting and the treatment for damaging the cell membrane, and measuring the number of viable cells and / or the cell viability by comparing the two intensities. A method for evaluating the toxicity of a substance. [25]. (i) applying a substance required for toxicity evaluation to cells, and (ii) applying a nucleic acid fluorescent stain for staining only dead cells to the obtained measurement sample, and measuring the intensity of the resulting fluorescence. (Iii) The toxicity evaluation method according to the above [24], wherein a treatment for damaging the cell membrane of the sample is performed, and the intensity of the fluorescence enhanced by killing the cells is measured. [26]. (i) applying the substance required for toxicity evaluation to the cells, (ii) applying a nucleic acid fluorescent dye for staining only dead cells to the measurement sample, and measuring the intensity of the fluorescence emitted by the substance, (iii) Another nucleic acid fluorescence stain is applied to another measurement sample and the cell membrane is damaged, and the intensity of the fluorescence emitted from the sample is measured [2].
4] The toxicity evaluation method described above.

[0028]

The present invention will be described in more detail with reference to the following Examples, which do not limit the present invention.

Example 1 (Measurement of Cell Survival Rate) (1) Method and Results MOLT-4 cells (human acute lymphoblastic leukemia cells: obtained from Osaka University Microorganisms Research Institute) at 37 ° C., 5% carbon dioxide, The cells were cultured on a petri dish under saturated steam conditions.
The culture solution was RPMI solution containing 10% fetal calf serum (GIBC
No. O, No. 31800-071; hereinafter, referred to as a culture solution). When the cell concentration became high, the sample on the Petri dish where the culture medium was changed appropriately (sample under good culture conditions) and the two samples on the Petri dish where culture was performed without changing the culture medium (Poor culture conditions) (Samples (1) and (2)) were used as cell viability measurement samples. Each sample was collected from a petri dish, placed in a cuvette for fluorescence measurement, and the following measurement was performed while stirring with a rotary stirrer. The fluorescence intensity was measured using a spectrofluorometer (Hitachi F
-4000 type) under the measurement conditions of an excitation wavelength of 420 nm and a fluorescence emission wavelength of 460 nm. First, PO-PRO-1 Iodide (trade name; M
No.5, manufactured by Molecular Probes. P-35
81) was added (final concentration 2.5 μM) and Fd ′ was measured. Subsequently, Triton X-100 (trade name; manufactured by PIERCE CHEMICAL) was added to the same measurement system (final concentration: 0.05%), Ft ′ was measured, and the cell viability was calculated by the above-described formula, and the results were obtained. The results are shown in Table 1. For comparison, the cell viability of each sample by the trypan blue staining method was measured under a microscope, and the results are shown in Table 1. The values measured by the trypan blue staining method were almost the same as the values measured in Example 1, indicating that the method according to the present invention was sufficiently applicable to the measurement of cell viability. In addition,
This measurement can be performed even if it is automated by the apparatus shown in FIG.

[0030]

[Table 1]

Example 2 (Quantitative Characterization of Reaction between Nucleic Acid Fluorescent Stain and Cell) (1) Method and Results MOLT-4, HL-60 (human promyelocytic leukemia cell), U937 (human histiocytic lymphoma cell) ), HT-
1080 (human fibrosarcoma cells) was used as a sample for measurement. Except for MOLT-4, they were obtained from Dainippon Pharmaceutical. Dilute the cell concentration of the sample for measurement 2 times with the culture solution,
A cell suspension was prepared and dispensed at 180 μl into each well of a 96-well microplate. 10 μl per well of PO-PRO-1 Iodi
de (trade name) was added (final concentration 2.5 μM), and 10 μl of Triton X-100 (trade name) was added to each well.
Was added (final concentration 0.05%), and the mixture was stirred for about 30 seconds with a plate stirrer, and then the fluorescence was measured. The results are shown in FIG. 3 and FIG.

FIG. 3 shows that there is a proportional relationship between the number of floating cells and the fluorescence intensity. HL-60
And U-937 have a cell number of 1.25 × 10 ⁴ to 4 × 1
0 proportional relationship between the cell number and fluorescence intensity in ^five ranges. MOLT-4 has a cell count of 1.25 × 10 ⁴
There is a proportional relationship between the number of cells and the fluorescence intensity within the range of 数 2 × 10 ⁵ cells.

FIG. 4 shows that there is a proportional relationship between the number of adherent cells and the fluorescence intensity. HT-10
80 has a proportional relationship between the number of cells and the fluorescence intensity within the range of 300 to 1 × 10 ⁵ cells. The fluorescence intensity was measured at an excitation wavelength of 420 nm and a fluorescence emission wavelength of 460 nm.
The measurement was carried out with an MTP-32 corona microplate reader (manufactured by Corona Electric Co., Ltd.) under the measurement conditions described above. This measurement can be performed even if it is automated by the apparatus shown in FIG.

(2) Discussion From the results shown in FIGS. 3 and 4, although it is necessary to measure the fluorescence intensity in a different measurement range for the floating cells and the adherent cells, the difference between the number of cells and the fluorescence intensity was measured. Since there is a proportional relationship, it was found that the present method can accurately measure the number of viable cells.

Example 3 (Evaluation of cytotoxicity due to drug) (1) Method and results Mitomycin C (manufactured by Kyowa Hakko Kogyo Co., Ltd.), which is an anticancer agent, was diluted with a culture solution, and 50 μl per well was added to a 96-well microplate. Dispensed. Subsequently, MOLT-4 cells were added at 3 × 10 5 per well.
^Four tubes / 150 μl were dispensed. Each well was cultured for 2 days under conditions of 37 ° C., 5% carbon dioxide, and saturated steam, and the following measurements were performed. In addition, the fluorescence measurement used the MTP-32 fluorometer (made by Corona Electric). 10 μl per well of PO-PRO-1 Iodi
de (trade name) was added (final concentration 2.5 μM), and the mixture was stirred for about 30 seconds with a plate stirrer.
d 'was determined. Further, 10 μl of Triton X-100 (trade name) was added to each well (final concentration: 0.05%), and the mixture was stirred for about 30 seconds with a plate stirrer, and fluorescence was measured to determine Ft ′. The number of viable cells and the cell viability were calculated by the above-described formulas, and the results are shown in Table 2. For comparison, Cancer Research was used for each sample.
Ch. 47, 936-942, 1987, the number of viable cells was measured by the MTT assay method, and the results are shown in Table 2. Table 3 shows the outline of the method of the present invention and the steps for measuring the number of viable cells by the MTT assay. Note that the above-described measurement according to the present invention can be performed by automation using the apparatus shown in FIG.

[0036]

[Table 2]

[0037]

[Table 3]

(2) Discussion As shown in Table 2, the cytotoxicity of mitomycin C was calculated to be 2.2 μg / ml in the method according to the present invention. This value is only on the order of μg / ml when compared with the value measured by the MTT method (5.5 μg / ml) which is currently most widely used for evaluating the cytotoxicity of a drug, and a numerical difference acceptable in the art. Met. From the above, it has been found that the method according to the present invention can be sufficiently applied to the evaluation of cytotoxicity by drugs. Further, the present invention has the advantage that the cell viability at each drug concentration can be calculated simultaneously, and that a great deal of information can be obtained in evaluating the drug efficacy. on the other hand,
As shown in Table 3, the time required for the measurement was completed in about 5 minutes in the method according to the present invention, and the time was dramatically reduced as compared with the existing MTT method (3 hours 20 minutes). In terms of operability, the method according to the present invention does not require centrifugation, washing, and dissolution of a dye, thus further increasing convenience.

Example 4 (Damage of Cell Membrane by Ultrasound) (1) Method and Results A 40 ml culture solution containing 5 × 10 ⁶ MOLT-4 cells was taken as a measurement sample into a cuvette for fluorescence measurement,
Ultrasonic crusher (SONICATOR ULTRASON)
IC PROCESSOR, Model W-225,
Serial No. G6453, 20kHz, HEA
T SYSTEMS-ULTRASONIC IN
C. ) For 10 seconds (crush strength level 3). The following measurement was performed while stirring the sample in the cuvette subjected to the ultrasonic treatment with a rotary stirrer. The fluorescence intensity was measured using a spectrofluorometer (Hitachi F-4000 type) under the conditions of an excitation wavelength of 420 nm and a fluorescence emission wavelength of 460 nm. First, PO-PRO-1 Iodide (trade name; M
No.5, manufactured by Molecular Probes. P-35
81) was added (final concentration 2.5 μM), and the fluorescence intensity was measured. As a result, it was 410 (the background fluorescence intensity was 10).

[0040]

According to the present invention, the number of viable cells and / or cell viability of a large number of specimens can be easily and rapidly increased by using a nucleic acid fluorescent dye for staining only dead cells and an agent for damaging cell membranes. Can be measured accurately,
It can be used to evaluate cytotoxicity of pesticides, cosmetics, foods, etc. In addition, its cytotoxicity can be easily and quickly evaluated.

[Brief description of the drawings]

FIG. 1 is a diagram showing an example of a viable cell count measuring device.

FIG. 2 is a diagram showing another example of a viable cell count measuring device.

FIG. 3 is a diagram showing the relationship between the number of planktonic cells and the fluorescence intensity.

FIG. 4 is a diagram showing the relationship between the number of adherent cells and the fluorescence intensity.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Drug which damages cell membrane 2 Drug injector 3 Nucleic acid fluorescent staining which stains only dead cells 4, 12 Polarizer 5, 9, 11 Lens 6 Measurement cuvette with stirrer 7 Device for converting analog signal to digital signal 8 Control system Reference Signs List 10 light source 13 photomultiplier tube 14 excitation side spectroscope 15 fluorescence spectrometer 16 96-well microplate with plate agitator 21 cell suspension 22 dispenser

Claims (12)

[Claims] 1. The intensity of fluorescence emitted from a measurement sample to which a nucleic acid fluorescent stain that stains only dead cells is applied, and the measurement sample subjected to the treatment with the nucleic acid fluorescent stain and the treatment to damage the cell membrane are emitted. A method for measuring the number of viable cells and / or the cell viability, wherein the respective intensities of fluorescence are measured and both intensities are compared. (2) Fluorescent nucleic acid stain for staining only dead cells is allowed to act on the measurement sample, and the intensity of the resulting fluorescence is measured. (Ii) Next, the cell membrane of the sample is damaged. The method according to claim 1, wherein the treatment is performed to measure the intensity of the fluorescence enhanced by killing the cells.
The measurement method described. (3) Activating a nucleic acid fluorescent dye which stains only dead cells on the measurement sample, measure the intensity of the fluorescence emitted from the dye, and (ii) fluorescently stain the nucleic acid with another measurement sample. 2. The method according to claim 1, wherein a treatment with an agent and a treatment to damage a cell membrane are performed, and the intensity of the fluorescence emitted from the treatment is measured. 4. The method according to claim 1, wherein the nucleic acid fluorescent stain is a cationic nucleic acid fluorescent stain. 5. The method according to claim 1, wherein the nucleic acid fluorescent dye is a ethidium homodimer or a cyanine nucleic acid fluorescent dye that stains only dead cells. 6. The method according to claim 1, wherein the cell membrane is damaged using an agent that damages the cell membrane. 7. The method according to claim 6, wherein the agent that damages the cell membrane is a surfactant. 8. The method according to claim 6, wherein the agent that damages the cell membrane is a polyoxyethylene type nonionic surfactant or an alcohol type nonionic surfactant. 9. The intensity of fluorescence emitted from a measurement sample to which a nucleic acid fluorescent dye that stains only dead cells has acted, and the measurement sample having undergone the treatment with the nucleic acid fluorescent stain and the treatment to damage the cell membrane. The method according to any one of claims 1 to 8, wherein each operation in a series of steps comprising measuring the fluorescence intensity and comparing the two intensities is performed by an automated device. 10. The intensity of fluorescence emitted from a measurement sample to which a nucleic acid fluorescent dye that stains only dead cells has acted, and the intensity of fluorescence emitted from the measurement sample to which the stain and an agent that damages the cell membrane have acted. The number of viable cells consisting of a nucleic acid fluorescent stain that stains only dead cells and an agent that damages cell membranes for measuring the number of viable cells and / or cell viability by comparing the two intensities, And / or a kit for measuring cell viability. (11) a means for supplying a nucleic acid fluorescent stain for staining only dead cells to a sample to be measured, and (b) measuring the intensity of fluorescence emitted from the sample to be measured which has been acted on by the nucleic acid fluorescent stain. Means, (c) means for damaging cell membranes, (d) intensity of fluorescence emitted by a sample to be measured which has been treated with a nucleic acid fluorescent dye which stains only dead cells and which has been treated to damage cell membranes. A number of viable cells and / or a means for comparing the intensities of the above (b) and (d) with respect to each of the measured fluorescence intensities. Cell viability measuring device. 12. A device for holding a drug that damages the cell membrane, (b) a device for holding a nucleic acid fluorescent dye that stains only dead cells, and (c) a device from the above device (a) or (b). The measuring apparatus according to claim 11, further comprising: a drug injector for supplying a drug to the sample to be measured, (d) a container capable of holding the sample to be measured, and (e) a spectrofluorometer.