CN113846145A

CN113846145A - Fluorescent dye stabilizer, kit containing same and related application

Info

Publication number: CN113846145A
Application number: CN202110962468.9A
Authority: CN
Inventors: 李彦萍
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-08-20
Filing date: 2021-08-20
Publication date: 2021-12-28

Abstract

The present invention provides a fluorescent dye stabilizer, a kit containing the same and related applications. The fluorescent dye stabilizer includes: triethylenediamine and n-propyl gallate, and the mass ratio of triethylenediamine and n-propyl gallate is 15:1-3.6. The stabilizer can improve the stability of fluorescent dyes. The problem that the fluorescent dye of necrotic cells in the prior art is poor in stability and easy to change colorless under high temperature or ethanol environment is solved, and it is suitable for the field of cell detection.

Description

Fluorescent dye stabilizer, kit containing same and related application

Technical Field

The invention relates to the field of cell detection, in particular to a fluorescent dye stabilizer, a kit containing the fluorescent dye stabilizer and related application.

Background

The great clinical significance of detecting circulating tumor cells CTC which are dropped from solid tumors into blood in the process of preventing and treating tumors is widely proved and reported by doctors in the tumor kingdom at home and abroad. The combined detection of tumor marker protein expression and chromosome number abnormality on CTC by applying a tumor protein immunofluorescence staining-chromosome fluorescence in situ hybridization (iFISH) method has become a main technical means for detecting CTC. Among them, FISH method for detecting chromosome number abnormality (heteroploidy) which is the most important feature of tumor cells is the most important.

At present, the clinical detection of the number of CTCs in a tumor patient in the treatment process is generally implemented for rapidly evaluating the curative effect and monitoring the generation of tumor drug resistance in real time. One important issue that continues to plague the vast majority of physicians is determining whether these detected CTCs are viable cells or dead cells that have been killed by the therapeutic drug. Compared with the method for detecting the number of CTCs of a patient who receives treatment of a plurality of courses of treatment to evaluate the curative effect, the method for rapidly and effectively distinguishing the CTCs of the live cells from the CTCs of the dead cells after the treatment of the medicine can enable a doctor to immediately and accurately judge whether the treatment medicine is effective in killing tumor cells, and has great clinical significance for tumor patients and doctors.

Dead cells are generally distinguished by exclusion through DNA-bound fluorescent dyes, and the characteristic that the permeability of dead cell membranes is increased and DNA dyes can enter the dead cells is utilized. Commonly used fluorescent dyes include 7-amino-actinomycin D (7-AAD), Propidium Iodide (PI) or other necrotic cell marker fluorescent dyes. However, the most common characteristics of the dead cell fluorescent dyes are thermolabile and unstable in alcohol solution. The above-described ifash method for identifying CTCs requires pretreatment of the sample in alcohol and heating to 76 ℃ to melt the DNA. However, after these necessary experimental steps, either 7-AAD or PI had lost color and failed to effectively identify dead cell CTCs. Therefore, there is a need to develop a material and a corresponding detection method capable of protecting fluorescent dye of necrotic cells to effectively distinguish various abnormal or normal chromosome dead cells and living cells in tumor tissues or biological fluids detected by ifash, so as to meet the urgent need of clinical application.

Disclosure of Invention

The invention mainly aims to provide a fluorescent dye stabilizer, a kit containing the fluorescent dye stabilizer and related application, so as to solve the problem of poor stability of fluorescent dye of necrotic cells in the prior art.

In order to achieve the above object, according to one aspect of the present invention, there is provided a fluorescent dye stabilizer including: triethylene diamine and n-propyl gallate, wherein the mass ratio of the triethylene diamine to the n-propyl gallate in the fluorescent dye stabilizer is 15: 1-3.6.

Furthermore, the fluorescent dye stabilizer comprises a cell buffer solution, wherein the triethylene diamine and the n-propyl gallate are dissolved in the cell buffer solution; preferably, the cell buffer is selected from the group consisting of buffers which are isotonic with 0.9 wt% physiological saline at pH 7.2-7.4 and do not contain amino groups; preferably the cell buffer is selected from phosphate buffers; preferably, the volume mass ratio of the phosphate buffer to the triethylene diamine is 1mL: (12-17.2) mg; more preferably 1mL (15-17.2) mg.

According to a second aspect of the present invention, there is provided a dead cell detection reagent, comprising a dead cell fluorescent dye and a stabilizer for the dead cell fluorescent dye, wherein the stabilizer is the above fluorescent dye stabilizer.

Further, the dead cell fluorescent dye is a fluorescent dye capable of being combined with an amino group; preferably, the dead cell fluorescent dye comprises any one or more of: 7-AAD (7-amino-actinomycin D), PI (propidium iodide) or DAPI (4', 6-diamidino-2-phenylindole).

Further, the dead cell fluorescent dye and the fluorescent dye stabilizer are respectively and independently stored or placed;

further, the dead cell detection reagent is a mixed solution of a dead cell fluorescent dye and a fluorescent dye stabilizer.

Furthermore, in the dead cell detection reagent, the working concentration of the fluorescent dye for dead cells is 5-10 mu g/mL, and the working concentration of the fluorescent dye stabilizer is 1.45-3.56 mg/mL.

According to a third aspect of the present invention, there is provided a kit comprising the above dead cell detection reagent.

Further, the kit further comprises an immunofluorescence detection reagent; preferably, the immunofluorescence detection reagent is a fluorescence in situ hybridization related reagent; preferably, the immunofluorescence detection reagent comprises any one or more of the following marker antibodies: a tumor marker antibody, a leukocyte marker antibody, a vascular endothelial cell marker antibody, a stem cell marker antibody, a mesenchymal transition marker antibody, or an endometrial cell marker antibody; preferably, the above tumor marker antibody includes an antibody of a tumor marker derived from epithelium; preferably, the tumor marker of epithelial origin is selected from any one or more of: estrogen Receptor (ER), Progestogen Receptor (PR), PD-L1, HER2, CEA, CA125, CA19-9, EpCAM, vimentin (vimentin), cytokeratin (cytokerin), Claudin 18.0, Claudin 18.2, AFP, HE4, CD47, or CD 74; preferably, the leukocyte marker antibody is selected from any one or more of the following: anti-CD 3, anti-CD 45, anti-CD 50, anti-CD 69, anti-CD 84, or anti-CD 102; preferably, the vascular endothelial cell marker antibody is selected from any one or more of the following: anti-CD 31 or anti-CD 146; preferably, the stem cell marker antibody is selected from any one or more of: anti-CD 34, anti-CD 44, anti-CD 133, or anti-EpCAM; preferably, the mesenchymal transition marker antibody is selected from any one or more of the following: vimentin antibody (anti-Vimentin), epithelial cell cadherin antibody (anti-E-cadherin).

According to a fourth aspect of the present invention, there is provided a method of identifying a dead or alive state of a cell, the method comprising: and adding the dead cell detection reagent into a cell sample to be detected for detection.

Further, the cell sample to be detected is a cell sample derived from a tumor tissue or a cell sample derived from a biological body fluid; preferably, the cell sample derived from tumor tissue is derived from any one of the following solid tumors: lung cancer, cervical cancer, esophageal cancer, colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, renal cancer, bladder cancer, prostate cancer, liver cancer, melanoma, glioma; preferably, the biological fluid is selected from any one or more of: blood, bone marrow, urine, cerebrospinal fluid, pleural fluid, ascites, or lymph node aspirate; preferably, the cell sample to be tested is a cell suspension.

Furthermore, the cell sample to be tested is a sample of various nucleated cells in a biological fluid, including blood-derived tumor cells (such as lymphoma cells), non-blood-derived tumor cells, vascular endothelial cells, stem cells, fetal cells in blood, endometrial cells or blood-derived leukocytes; preferably, the blood-borne tumor cells are lymphoma cells; preferably, the non-blood derived tumor cell is a solid tumor cell; preferably, the stem cell is a tumor stem cell.

Further, the cell sample to be detected is an immune cell; preferably, the immune cell is a lymphocyte.

According to a fifth aspect of the present invention, there is provided a fluorescent in situ hybridization method, comprising: staining dead cells in a cell sample to be detected by using the dead cell detection reagent; and (4) carrying out chromosome fluorescence in situ hybridization on the stained cell sample to be detected.

Further, in the reagent for detecting dead cells, the fluorescent dye for dead cells and the fluorescent dye stabilizer are separately stored or placed, and the method comprises the following steps: adding a dead cell dye and an optional fluorescence labeling antibody into a fluorescent dye stabilizer to obtain a mixed solution; staining a cell sample to be detected by using the mixed solution to obtain stained cells; performing fluorescence in situ hybridization on the stained cells; preferably, the fluorescently labeled antibody comprises any one or more of the following marker antibodies: a tumor marker antibody, a leukocyte marker antibody, a vascular endothelial cell marker antibody, a stem cell marker antibody, a mesenchymal transition marker antibody, or an endometrial cell marker antibody; preferably, the tumor marker antibody comprises an antibody to a tumor marker of epithelial origin; preferably, the tumor markers of epithelial origin are selected from any one or more of: estrogen Receptor (ER), Progestogen Receptor (PR), PD-L1, HER2, CEA, CA125, CA19-9, EpCAM, vimentin (vimentin), cytokeratin (cytokerin), Claudin 18.0, Claudin 18.2, AFP, HE4, CD47, or CD 74; preferably, the leukocyte marker antibody is selected from any one or more of: anti-CD 3, anti-CD 45, anti-CD 50, anti-CD 69, anti-CD 84, or anti-CD 102; preferably, the vascular endothelial cell marker antibody is selected from any one or more of: anti-CD 31, anti-CD 146; preferably, the stem cell marker antibody is selected from any one or more of: anti-CD 34, anti-CD 44, anti-CD 133, anti-EpCAM; preferably, the mesenchymal transition marker antibody is selected from any one or more of: vimentin antibody (anti-Vimentin), epithelial cell cadherin antibody (anti-E-cadherin).

Further, the cell sample to be detected is a cell sample derived from a tumor tissue or a cell sample derived from a biological body fluid; preferably, the sample of tumor tissue-derived cells is derived from any one of the following solid tumors: lung cancer, cervical cancer, esophageal cancer, colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, renal cancer, bladder cancer, prostate cancer, liver cancer, melanoma, glioma; preferably, the biological fluid is selected from any one or more of: blood, bone marrow, urine, cerebrospinal fluid, pleural fluid, ascites, or lymph node aspirate; preferably, the cell sample to be tested is a cell suspension.

Further, the cell sample to be tested in the above method is circulating vascular endothelial cells, stem cells, fetal cells in blood, endometrial cells or blood-derived leukocytes; preferably, the stem cell is a tumor stem cell.

Further, the cell sample to be tested in the above method is an immune cell; preferably, the immune cell is a lymphocyte.

Further, the fluorescence in situ hybridization method comprises the following steps: adding the mixed solution into a cell suspension of a cell sample to be detected for dyeing to obtain a dyed cell; washing, smearing and chromosome fluorescence in-situ hybridization are carried out on the stained cells in sequence; preferably, a phosphate buffer is used for washing; preferably, washing is carried out in a mode of centrifuging for 3-5 minutes at the speed of 1000-1500 revolutions per minute; preferably, the number of flushing is 2-5.

Further, the fluorescence in situ hybridization of the chromosome comprises: carrying out gradient dehydration on the washed stained cells by adopting ethanol to obtain dehydrated cells; placing the dehydrated cells at 70-80 ℃ for DNA denaturation to obtain DNA denatured cells; performing fluorescence in situ hybridization on the DNA denatured cells by adopting a target fluorescence probe; preferably, the target fluorescent probe comprises a chromosome probe; preferably, the chromosome probe comprises any one or more of: a CEP7, CEP8 or ALK fusion gene probe or a hematological tumor probe; preferably, the hematologic tumor probe comprises a lymphoma probe.

According to a sixth aspect of the present invention, there is provided a use of the above fluorescent dye stabilizer or dead cell detection reagent or kit for detecting living cells and dead cells, which is an application for non-diagnostic purposes.

Further, the application comprises staining the dead cells with a necrotic cell detection reagent; preferably, the application further comprises, on the basis of staining dead cells: and (4) judging the state of the living cells by using a FISH or iFISH method.

The technical scheme of the invention provides the fluorescent dye stabilizer, the kit containing the fluorescent dye stabilizer and related applications, which are beneficial to protecting the fluorescent dye of necrotic cells to keep the fluorescent dye stable, and further can effectively distinguish various abnormal or normal dead cells and living cells of chromosomes in biological body fluid or cell suspension liquid derived from tumor tissues.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows a schematic diagram of single cell discrimination at different fluorescence according to example 1 of the present invention, wherein,

the 7-AAD + fluorescent stabilizer is marked as dead cells; CD45 labeled leukocytes; FISH CEP8 is labeled as a chromosome of the cell; DAPI labeled as nuclei; the combined images are labeled as discriminatory images of live and dead cells after staining and whether they are tumor cells or not.

FIG. 2 shows a schematic diagram of single cell discrimination at different fluorescence according to example 2 of the present invention, wherein,

the PI + fluorescent stabilizer is marked as dead cells; HER2 labeled tumor cells; FISH CEP8 is labeled as a chromosome of the cell; DAPI labeled as nuclei; the combined images are labeled as discriminatory images of live and dead cells after staining and whether they are tumor cells or not.

FIG. 3 shows a schematic diagram of single cell discrimination at different fluorescence according to example 3 of the present invention, wherein,

the PI + fluorescent stabilizer is marked as dead cells; CD45 labeled leukocytes; FISH CEP8 is labeled as a chromosome of the cell; DAPI labeled as nuclei; the combined images are labeled as discriminatory images of live and dead cells after staining and whether they are tumor cells or not.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

Interpretation of terms:

FISH: the chromosome fluorescence in situ hybridization technique is a technique which adopts specific single-stranded nucleic acid with a known sequence as a probe, labels biotin or fluorescein, leads DNA-DNA to be hybridized in situ by a base complementary pairing rule at a certain temperature and ion concentration, displays by a fluorescence method, and finally labels the DNA (such as chromosome number) at the original position of a monolayer cell slide and a tissue section (such as a paraffin section or a frozen section).

iFISH: namely, the immunofluorescence antibody staining-chromosome fluorescence in situ hybridization technique is an integration technique which can simultaneously carry out immunofluorescence antibody staining and FISH on the same cells.

Cell buffer: a solution capable of maintaining the osmotic pressure and pH of the solution equal to the osmotic pressure and pH of the cells when a small amount of acid or base is added.

Tumor markers: a characteristic marker protein on tumor cells.

Tumor marker antibodies: monoclonal or polyclonal antibodies specific for tumor markers.

Cell sample: corresponding to the tissue sample, the term "sample" refers to a sample containing free cells, including various biological fluid samples containing free cells and cell suspension samples prepared from the tissue sample.

As mentioned in the background art, the existing fluorescent dye for necrotic cells is unstable and colorless under the action of high temperature or ethanol, so that the dead cells cannot be effectively identified, and the purpose of effectively distinguishing various abnormal or normal chromosome dead cells and living cells in tumor tissues or biological body fluid cannot be achieved.

Therefore, the present inventors have conducted intensive studies on fluorescent dye stabilizers and have found a fluorescent dye stabilizer which can stabilize and maintain the existing fluorescent dyes for necrotic cells under high temperature or the action of ethanol. Thus a series of protection schemes of the present application are proposed.

In one embodiment, the present invention provides a fluorescent dye stabilizer comprising: triethylene diamine and n-propyl gallate, wherein the mass ratio of the triethylene diamine to the n-propyl gallate in the fluorescent dye stabilizer is 15: 1-3.6.

Triethylene Diamine (TEDA or DABCO), molecular weight 112.18, molecular formula C₆H₁₂N₂Mainly used for catalyzing ethylene polymerization and ethylene oxide polymerization in the industry; n-propyl gallate (NPG), molecular weight 212.2, molecular formula C₁₀H₁₂O₅Is stable to heat and is mainly used as food antioxidant. The invention discovers that the two compounds have the function of protecting the fluorescent dye after being mixed according to a proper proportion, and can ensure that the existing fluorescent dye for necrotic cells is stable and does not fade under the action of high temperature or ethanol. The mass ratio of the triethylene diamine to the n-propyl gallate is 15: 1-3.6; the mass ratio can be optimized according to subsequent experiments so as to ensure that the fluorescent staining of dead cells does not influence the presentation of other subsequent experimental results, and the mass ratio of the triethylene diamine to the n-propyl gallate is preferably 15: 1-1.22.

In the fluorescent dye stabilizer, the fluorescent dye stabilizer also comprises a cell buffer solution, and the triethylene diamine and the n-propyl gallate can be dissolved in the cell buffer solution; preferably, the cell buffer is selected from the group consisting of buffers which are isotonic with 0.9 wt% physiological saline at pH 7.2-7.4 and do not contain an amino group; more preferably, the cell buffer is selected from phosphate buffers; preferably, the volume mass ratio of the phosphate buffer to the triethylene diamine is 1mL: (12-17.2) mg; more preferably 1mL (15-17.2) mg.

The triethylene diamine and the n-propyl gallate can be dissolved in a cell buffer solution to form a fluorescent dye stabilizer. The cell buffer solution is a solution which can keep the osmotic pressure and the pH value of the solution equal to those in cells when a small amount of acid or alkali is added, and the cell buffer solution is used as the solution, so that the fluorescent dye stabilizer can be ensured not to damage living cells due to osmotic pressure, pH and the like to influence the accuracy of an experimental result when in use. Therefore, a buffer solution, a minimal medium, or the like, which is generally used to maintain the state of cell activity, is used in the present application. For example, phosphate buffer, physiological saline, etc. The ratio of the most commonly used cell buffer, i.e., phosphate buffer to triethylenediamine, is preferably selected so that a fluorescent dye stabilizer that is easy to produce and use can be obtained at concentrations above which can cause cell swelling due to hypotonic interactions or shrinkage due to hypertonic interactions (water within the cell is pumped out of the cell).

In a second embodiment of the present invention, a reagent for detecting dead cells is provided, the reagent for detecting dead cells comprises a fluorescent dye for dead cells and a stabilizer for the fluorescent dye for dead cells, wherein the stabilizer is the fluorescent dye stabilizer.

The identification of dead cells generally utilizes the characteristic that DNA can be combined with fluorescent dye of dead cells, the cell membrane permeability of the dead cells is increased, and the fluorescent dye of the dead cells can enter and dye the DNA; and the cell membrane permeability of the living cells is small, and the fluorescent dye of the dead cells cannot enter, so that the DNA of the living cells cannot be stained. By utilizing the characteristics, dead cells and living cells can be distinguished conveniently and rapidly.

However, the commonly used dead cell fluorescent dyes at present are PI, 7-AAD, DAPI and the like, and the dead cell fluorescent dyes have the greatest common characteristics of thermolabile property and instability in an ethanol solution, so that the dead cell fluorescent dyes can not be used in a subsequent experiment with a heating step or ethanol treatment, and the application of the dead cell fluorescent dyes is greatly limited. The dead cell detection reagent provided by the embodiment comprises a dead cell fluorescent dye and a stabilizer of the dead cell fluorescent dye, can solve the influence of temperature or ethanol on the stability of the dead cell fluorescent dye, and greatly increases the application range of the dead cell fluorescent dye.

In the above dead cell detection reagent, the dead cell fluorescent dye includes, but is not limited to, any one or more of the following: 7-AAD, PI, or DAPI.

In the dead cell detection reagent, the dead cell fluorescent dye and the fluorescent dye stabilizer are respectively and independently stored or placed.

In the above dead cell detection reagent, the detection reagent is a mixed solution of a dead cell fluorescent dye and a fluorescent dye stabilizer.

In the dead cell detection reagent, the working concentration of the fluorescent dye for dead cells is 5-10 micrograms/mL, and the working concentration of the fluorescent dye stabilizer is 1.45-3.56 mg/mL.

The fluorescent dye stabilizer in the dead cell detection reagent can protect various dead cell fluorescent dyes, can be used in combination with different dead cell fluorescent dyes, and meets the requirements of different detections and experiments. Dead cell fluorescent dyes include, but are not limited to, any one or more of the following: 7-AAD, PI or DAPI. Among them, DAPI belongs to a dead cell dye reagent, but one generally does not use this dye to distinguish between live cells and dead cells, but adds DAPI in the last step of the experiment to stain all dead cells as a uniform recognition standard for all cells, and counts the number of all cells. During production, transportation and storage, the dead cell fluorescent dye and the fluorescent dye stabilizer can be separately stored or placed respectively so as to improve the stability of the two reagents; and when the reagent is used, the two reagents are mixed according to a certain proportion, so that detection personnel can conveniently adjust the reagent according to the difference of specific experimental materials, and can select proper concentration within an optimal working concentration range for detection. The two can also be directly prepared into a finished product form according to the concentration required by the work, so that the mixing before the operation is avoided, and the direct use is convenient. In the mother liquor of the fluorescent dye and the fluorescent dye stabilizer, the total concentration of triethylene diamine and n-propyl gallate used as the stabilizer can be 16-21.33 mg/mL, when the fluorescent dye and the fluorescent dye stabilizer are used, a certain volume of the mother liquor is added into a sample solution with the volume 5-10 times that of the mother liquor, the operation is convenient, the mother liquor is easy to mix uniformly, and the fluorescent dye stabilizer are diluted to the working concentration of 1.45-3.56 mg/mL, so that the fluorescent dye stabilizer can play a role in protection.

In a third embodiment of the present invention, a kit is provided, which comprises the above dead cell detection reagent.

In the kit, an immunofluorescence detection reagent is included; preferably, the immunofluorescence detection reagent may be a fluorescence in situ hybridization-related reagent; preferably, the immunofluorescence detection reagent comprises any one or more of the following marker antibodies: a tumor marker antibody, a leukocyte marker antibody, a vascular endothelial cell marker antibody, a stem cell marker antibody, a mesenchymal transition marker antibody, or an endometrial cell marker antibody; preferably, the tumor marker antibody comprises an antibody to a tumor marker of epithelial origin; preferably, the tumor markers of epithelial origin are selected from any one or more of: ER, PR, PD-L1, HER2, CEA, CA125, CA19-9, EpCAM, Vimentin, cytokeratin, Claudin 18.0, Claudin 18.2, AFP, HE4, CD47 or CD 74; preferably, the leukocyte marker antibody is selected from any one or more of: anti-CD 3, anti-CD 45, anti-CD 50, anti-CD 69, anti-CD 84, or anti-CD 102; preferably, the vascular endothelial cell marker antibody is selected from any one or more of: anti-CD 31 or anti-CD 146; preferably, the stem cell marker antibody is selected from any one or more of: anti-CD 34, anti-CD 44, anti-CD 133, or anti-EpCAM; preferably, the mesenchymal transition marker antibody is selected from any one or more of: anti-Vimentin, anti-E-cadherin.

The reagent comprises the dead cell detection reagent, can develop color under the influence of high temperature or ethanol, and plays a role in identifying live cells and dead cells. The kit may further comprise immunofluorescence detection reagents, such as fluorescence in situ hybridization related reagents, for performing Fluorescence In Situ Hybridization (FISH) experiments, using antibodies with different specificities for detecting cell samples.

In a fourth embodiment of the present invention, there is provided a method for identifying a dead or alive state of a cell, the method comprising: and adding the dead cell detection reagent into a cell sample to be detected for detection.

In the above method, the cell sample to be tested may be a cell sample derived from a tumor tissue or a cell sample derived from a biological fluid; preferably, the tumor tissue-derived cell sample includes, but is not limited to, any one of the following solid tumors: lung cancer, cervical cancer, esophageal cancer, colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, renal cancer, bladder cancer, prostate cancer, liver cancer, melanoma, glioma; biological fluids include, but are not limited to, any one or more of the following: blood, bone marrow, urine, cerebrospinal fluid, pleural fluid, ascites, or lymph node aspirate; the cell sample to be tested may also be a cell suspension.

In the above method, the cell sample to be tested includes, but is not limited to, blood-derived tumor cells (e.g., lymphoma cells), non-blood-derived tumor cells, vascular endothelial cells, stem cells, blood fetal cells, endometrial cells, or blood-derived leukocytes; preferably, the stem cells may be tumor stem cells.

In the above method, the cell sample to be tested may be an immune cell; preferably, the immune cell may be a lymphocyte.

The method uses the dead cell detection reagent to dye a cell sample, and uses the difference of cell membrane permeability of live cells and dead cell fluorescent dye to dye DNA of the dead cells, thereby identifying the survival state of the cells in the cell sample. The method has effects on various cells, such as cells derived from tumor tissue and cells derived from biological fluid, and has wide application range.

According to the different specific sources and types of the cell samples to be detected, the survival state of the cell samples can be detected, and on the basis, other indexes or performance conditions can be further detected according to the different research purposes. Accordingly, in a fifth embodiment of the present invention, there is provided a fluorescent in situ hybridization method, comprising: and dyeing dead cells in the cell sample to be detected by using the dead cell detection reagent, and carrying out fluorescence in-situ hybridization on the dyed cell sample to be detected.

The fluorescence in situ hybridization method comprises the steps of firstly, carrying out fluorescence dyeing on a cell sample by using the dead cell detection reagent containing the dead cell fluorescent dye and the fluorescent dye stabilizer, and protecting the stability of the dead cell fluorescent dye. And (3) distinguishing live cells and dead cells in the cell sample by using the difference of cell membrane permeability of the live cells and the dead cells.

In the above method, when the dead cell fluorescent dye and the fluorescent dye stabilizer in the dead cell detection reagent are separately stored or left to stand in the step of staining the cells, the method comprises: adding a dead cell dye and an optional fluorescence labeling antibody into a fluorescent dye stabilizer to obtain a mixed solution; staining a cell sample to be detected by using the mixed solution to obtain stained cells; performing fluorescence in situ hybridization on the stained cells; preferably, the fluorescently labeled antibody comprises any one or more of the following marker antibodies: a tumor marker antibody, a leukocyte marker antibody, a vascular endothelial cell marker antibody, a stem cell marker antibody, a mesenchymal transition marker antibody, or an endometrial cell marker antibody; preferably, the tumor marker antibody comprises an antibody to a tumor marker of epithelial origin; preferably, the tumor markers of epithelial origin are selected from any one or more of: ER, PR, PD-L1, HER2, CEA, CA125, CA19-9, EpCAM, Vimentin, cytokeratin, Claudin 18.0, Claudin 18.2, AFP, HE4, CD47 or CD 74; preferably, the leukocyte marker antibody is selected from any one or more of: anti-CD 3, anti-CD 45, anti-CD 50, anti-CD 69, anti-CD 84, or anti-CD 102; preferably, the vascular endothelial cell marker antibody is selected from any one or more of: anti-CD 31, anti-CD 146; preferably, the stem cell marker antibody is selected from any one or more of: anti-CD 34, anti-CD 44, anti-CD 133, anti-EpCAM; preferably, the mesenchymal transition marker antibody is selected from any one or more of: anti-Vimentin, anti-E-cadherin.

The method can identify the survival state of various cells such as cells from tumor tissues and cells from biological body fluid, and has wide application. The operation steps of specifically staining the cell sample to be detected are slightly different according to different storage or placement modes of the two components in the dead cell detection reagent. To further enhance the stabilizing effect of the fluorescent dye stabilizer on the dead cell fluorescent dye, the preferred embodiment described above employs freshly prepared dead cell detection reagents for staining.

The fluorescent-labeled antibody herein may be added or not appropriately selected depending on the purpose of actual research. For example, in the case where it is not necessary to determine the type of cells or whether there is chromosomal abnormality, the addition of a fluorescent-labeled antibody may be omitted. For another example, if it is required to detect whether the cell to be detected is a tumor cell, a fluorescence-labeled antibody of a tumor marker may be added, and then it may be determined whether the cell is a tumor cell according to whether the fluorescence-labeled antibody of a tumor marker emits light.

Therefore, in some preferred embodiments, the cell sample to be tested can be a cell sample derived from tumor tissue or a cell sample derived from biological fluid; preferably, the tumor tissue-derived cell sample includes, but is not limited to, any one of the following solid tumors: lung cancer, cervical cancer, esophageal cancer, colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, renal cancer, bladder cancer, prostate cancer, liver cancer, melanoma, glioma; biological fluids include, but are not limited to, any one or more of the following: blood, bone marrow, urine, cerebrospinal fluid, pleural fluid, ascites, or lymph node aspirate; when the cell sample to be tested can be a cell sample derived from tumor tissue, the cell sample to be tested is a cell suspension.

In other preferred embodiments, the test cell sample includes, but is not limited to, a sample of various nucleated cells in a biological fluid, including blood-borne tumor cells, non-blood-borne tumor cells, circulating vascular endothelial cells, stem cells, blood fetal cells, endometrial cells, or blood-borne leukocytes; preferably, the blood-borne tumor cells are lymphoma cells; preferably, the non-blood derived tumor cell is a solid tumor cell; preferably, the stem cell may be a tumor stem cell.

In certain preferred embodiments, the cell sample to be tested is an immune cell; preferably, the immune cell may be a lymphocyte.

In a preferred embodiment, the method comprises: adding the mixed solution into a cell suspension of a cell sample to be detected for dyeing to obtain a dyed cell; washing, smearing and fluorescence in situ hybridization are carried out on the stained cells in sequence; preferably, the washing can be performed with a phosphate buffer; washing by centrifuging for 3-5 minutes at the speed of 1000-1500 rpm; the number of washing times can be 2-5.

In a preferred embodiment, the fluorescence in situ hybridization comprises: carrying out gradient dehydration on the washed stained cells by adopting ethanol to obtain dehydrated cells; placing the dehydrated cells at 70-80 ℃ for DNA denaturation to obtain DNA denatured cells; performing fluorescence in situ hybridization on the DNA denatured cells by adopting a target fluorescence probe; preferably, the target fluorescent probe includes, but is not limited to, any one or more of the following: various chromosome probes: CEP7, CEP8, ALK fusion gene probes, or hematologic tumor (e.g., lymphoma) probes.

After the dead cell detection reagent is used, the cell sample is cleaned, and the uncolored dead cell fluorescent dye is eluted, so that the interference on the subsequent experiment is prevented. Dehydrating the dyed cells, heating to denature DNA, opening double helix chains of the DNA to form 2 single-stranded DNAs, adding a target fluorescent probe, and complementarily pairing a specific nucleotide sequence on the target fluorescent probe and a base on the single-stranded DNA at room temperature to complete fluorescent in-situ hybridization. The DNA with the fluorescent label can be observed under a fluorescent microscope, thereby determining the number of chromosomes, judging whether the number of the chromosomes is abnormal or not and judging whether the cell is possible to be a tumor cell or not. In this fluorescence in situ hybridization method, DNA is denatured by heating, and cells are dehydrated by ethanol, and the fluorescent dye for dead cells used before does not fade in the presence of the fluorescent dye stabilizer, so that it is possible to determine whether tumor cells in a cell sample are live cells or dead cells.

In the fluorescence in situ hybridization method, fluorescence labeling antibodies, such as leucocyte marker antibodies and tumor marker antibodies, can also be selectively added, so that whether the cells are blood-borne cells or specific tumor cells can be judged, and the cells can be more accurately judged.

In a sixth embodiment of the present invention, there is provided a use of the above fluorescent dye stabilizer, dead cell detection reagent or kit for detecting living cells and dead cells.

In the above application, the application comprises staining dead cells with a necrotic cell detection reagent; preferably, the application further comprises, on the basis of staining dead cells: and (4) judging the state of the living cells by using a FISH or iFISH method.

The application utilizes the fluorescent dye stabilizer, the dead cell detection reagent or the kit, can judge the survival state of the cell, simultaneously protect the fluorescent dye of the dead cell from being damaged by high temperature and ethanol in subsequent experiments, for example, FISH or iFISH tests can be carried out on the cell after dyeing to determine the number of chromosomes of the cell, and the identification efficiency of the cell state is improved. At present, the iFISH method is generally used clinically to detect the quantity of CTCs in the treatment process of tumor patients, and is used for rapidly evaluating the curative effect and monitoring the generation of tumor drug resistance in real time. Subject to the limitations of previous detection conditions, the dead cell fluorescent dye retains color under the experimental conditions of ifash, and thus there is no way to distinguish whether the detected CTCs are live cells or dead cells that have been killed by the therapeutic drug. By utilizing the application, the clinical problem can be solved, and the CTC of the living cells and the CTC of the dead cells after the drug treatment can be effectively and quickly distinguished.

The advantageous effects of the present application will be explained in further detail below with reference to specific examples.

Example 1: detection of circulating tumor cells CTC in blood from patients with lung cancer to assess efficacy

A "fluorescent stabilizer" working stock was prepared containing 15mg of DABCO and 1mg of NPG per ml of phosphate buffered saline (PBS, pH 7.4).

Preparing a 'fluorescent stabilizer working solution': to 0.1mL of "fluorescent stabilizer stock" was added 20. mu.L (10. mu.g) of 7-AAD and a fluorescent-labeled antibody (anti-leukocyte CD45 antibody), and gently and thoroughly mixed.

Preparing and dyeing a peripheral blood specimen: non-small cell lung cancer patients who received 2-way chemotherapy received 6mL of intravenous blood. Centrifugation was carried out at 1500 rpm for 3 minutes to remove supernatant plasma. The blood cells are placed on the top layer of a non-blood-derived cell centrifugation medium, and deposited red blood cells are removed by centrifugation. After the leukocytes were collected, 300. mu.l of immunomagnetic beads coupled with anti-leukocyte antibodies were added thereto, and gently mixed for 20 minutes. The leukocytes bound to the magnetic beads were removed using a magnetic holder. The enriched peripheral blood lung cancer Circulating Tumor Cells (CTCs) were resuspended in 1mL PBS (pH 7.4) to prepare a CTC cell suspension. Adding 0.1mL of the prepared 'fluorescent stabilizer working solution' into 1mL of CTC cell suspension, and fully and uniformly mixing. Incubate for 30 minutes at room temperature in the dark. Washing with PBS for 2 times, centrifuging for 5 minutes at 1000 rpm, collecting cells, smearing on a slide, drying, and performing subsequent FISH experiment.

FISH experiment: the main experimental steps are dehydration with 75% -100% absolute ethyl alcohol gradient, adding 8 # chromosome probe CEP8 for hybridization (high temperature melting DNA, room temperature hybridization 8 # chromosome probe), the hybridization time is 6 hours. Slides were washed, DAPI added, and all cells were stained to identify true cells.

Observing and analyzing a specimen: the staining of each channel was observed under a microscope (as described in FIG. 1).

And (3) cell discrimination: the FISH signal is diploid or non-diploid; PI is stained as dead cells and not as live cells; staining with or without anti-leukocyte CD45 antibody; nuclear DAPI positive in all cells.

And (3) detection results: the detection shows that after 2 courses of chemotherapy, 2 died lung cancer circulating tumor cells playing an important role in the tumor metastasis process in peripheral blood of the lung cancer patient, and 1 survived lung cancer circulating tumor cell, which indicates that the current treatment scheme is effective, and the patient should continue to maintain the current chemotherapy.

Example 2: DTC (diffuse tumor cell) for detecting necrotic disseminated tumor cells in lymph node puncture specimen of breast cancer patient to evaluate curative effect

Configuration of "fluorescent stabilizer mother liquor": each ml of phosphate buffered saline (PBS, pH7.4) contained 15mg of DABCO and 1mg of NPG.

Preparing a 'fluorescent stabilizer working solution': to 0.1mL of "fluorescent stabilizer stock solution" were added 20. mu.L of PI (5. mu.g) and a fluorescent-labeled antibody (anti-tumor marker antibody HER2), and the mixture was gently and thoroughly mixed.

Preparing and dyeing a lymph node puncture specimen: breast cancer patients were pathologically examined positive for HER 2. After the patient receives the targeted drug herceptin for 3 treatment courses, lymph node puncture is performed, and a 0.5mL lymph node puncture specimen is obtained. To 0.5mL lymph node puncture specimen was added 5mL PBS (pH 7.4) and washed once by centrifugation (1500 rpm, 1 min) to remove protein impurities. The supernatant was discarded to 0.5mL and gently resuspended. 0.1mL of the "working solution of fluorescent stabilizer" was mixed with 0.5mL of the cell suspension, and incubated for 30 minutes at room temperature in the dark. Washing with PBS for 2 times, centrifuging at 1000 rpm for 5 minutes, collecting cells, smearing on a slide, drying, and performing subsequent FISH experiment to detect chromosome characteristics of the cells.

Observing and analyzing a specimen: the staining of each channel was observed under a microscope (as described in FIG. 2).

And (3) cell discrimination: the FISH signal is diploid or non-diploid; PI is stained as dead cells and not as live cells; staining with or without anti-tumor marker antibodies; nuclear DAPI positive in all cells.

And (3) detection results: the detection shows that after the breast cancer advanced patient is treated by the targeted drug Hisex, one larger cell with 4 times of chromosome 8 is necrotic cell in the Dispersive Tumor Cell (DTC) transferred to lymph node, but HER2⁺The tumor cells are still living cells, which indicates that the existing treatment scheme uses the pure herceptin for treatment, the curative effect is not expected, and a chemotherapy means is required to be added to completely inactivate the tumor cells in the lymph nodes, so that the possibility of tumor distant metastasis is stopped as soon as possible.

Example 3: detecting necrotic disseminated tumor cells DTC in a bone marrow puncture specimen of a patient with lung cancer metastatic bone cancer to evaluate the curative effect and prepare a 'fluorescent stabilizer mother solution':

mother liquor-1: 17.2mg DABCO and 4.13mg NPG per ml phosphate buffered saline (PBS, pH7.4) (21.33mg/ml for treatment of specimens 24-72 hours after bone marrow harvest);

mother liquor-2: each ml of phosphate buffered saline (PBS, pH7.4) contained 15mg of DABCO and 1mg of NPG (16mg/ml, for processing samples within 24 hours after bone marrow harvest).

Preparing a 'fluorescent stabilizer working solution-1': to 0.1mL "fluorescent stabilizer stock-1" was added 20 microliters (6 micrograms) of PI along with a fluorescently labeled antibody (anti-tumor marker antibody HER 2);

preparing a 'fluorescent stabilizer working solution-2': to 0.1mL "fluorescent stabilizer stock-2" was added 20 microliters (3 micrograms) of PI along with a fluorescently labeled antibody (anti-tumor marker antibody HER 2).

The working solution is gently and fully mixed.

Preparing and dyeing a bone marrow puncture specimen: after receiving chemotherapy for 1 course of treatment, patients with advanced lung cancer bone metastasis undergo bone marrow puncture, and 1ml of bone marrow puncture specimens are obtained. The specimens were processed within 24 hours or stored in a refrigerator for less than 72 hours. To 0.5mL of the bone marrow aspirate was added 5mL of PBS (pH 7.4) and washed once by centrifugation (1500 rpm, 1 minute) to remove protein impurities. The supernatant was discarded to 0.5mL and gently resuspended. 0.1mL of the "fluorescent stabilizer working solution-1" or the "fluorescent stabilizer working solution-2" is mixed with 0.5mL of cell suspension, and the mixture is incubated for 30 minutes at room temperature in a dark place. Washing with PBS for 2 times, centrifuging at 1000 rpm for 5 minutes, collecting cells, smearing on a slide, drying, and performing subsequent FISH experiment to detect chromosome characteristics of the cells.

Observing and analyzing a specimen: the staining of each channel was observed under a microscope (as described in FIG. 3).

And (3) detection results: the detection shows that after a patient with the advanced lung cancer is treated by chemotherapy for one course of treatment, one No. 8 chromosome is 4 ploid, one tumor cell with 3 ploid is necrotic cell, and the other tumor cell with triploid is viable cell, so that the existing chemotherapy treatment scheme can achieve the expected effect, and the patient can maintain the existing chemotherapy scheme and continue chemotherapy.

From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the fluorescent dye stabilizer consisting of triethylene diamine and n-propyl gallate is found, and the fluorescent dye stabilizer is beneficial to solving the problems that the existing necrotic cell fluorescent dye is unstable and easy to fade at high temperature or under the action of ethanol; by utilizing the fluorescent dye stabilizer, the application conditions of the fluorescent dye for necrotic cells are expanded.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A fluorescent dye stabilizer, characterized in that, the fluorescent dye stabilizer comprises: triethylenediamine and n-propyl gallate, and the triethylenediamine and the gallic acid in the fluorescent dye stabilizer The mass ratio of n-propyl acid is 15:1～3.6.

2. The fluorescent dye stabilizer according to claim 1, wherein the fluorescent dye stabilizer further comprises a cell buffer, and the triethylenediamine and the n-propyl gallate are dissolved in the cell buffer in liquid;

Preferably, the cell buffer is selected from buffers with osmotic pressure and 0.9wt% isotonic saline, pH 7.2-7.4, and no amino groups, more preferably, the cell buffer is selected from phosphate buffers;

Preferably, the volume-to-mass ratio of the phosphate buffer to the triethylenediamine is 1 mL: (12-17.2) mg, more preferably 1 mL: (15-17.2) mg.

3. A dead cell detection reagent, wherein the detection reagent comprises a dead cell fluorescent dye and a stabilizer of the dead cell fluorescent dye, wherein the stabilizer is the fluorescent dye according to claim 1 or 2 stabilizer.

4. The dead cell detection reagent according to claim 3, wherein the dead cell fluorescent dye is a fluorescent dye that can be combined with an amino group; preferably, the dead cell fluorescent dye comprises any one of the following or Various: 7-AAD, PI or DAPI.

5 . The dead cell detection reagent according to claim 3 , wherein the dead cell fluorescent dye and the fluorescent dye stabilizer are placed or stored separately. 6 .

6 . The dead cell detection reagent according to claim 3 , wherein the detection reagent is a mixed solution of the dead cell fluorescent dye and the fluorescent dye stabilizer. 7 .

The dead cell detection reagent according to claim 5 or 6, characterized in that, in the detection reagent, the working concentration of the dead cell fluorescent dye is 5-10 μg/mL, and the working concentration of the fluorescent dye stabilizer is 5-10 μg/mL. The concentration is 1.45～3.56mg/mL.

8. A kit, characterized in that the kit comprises the dead cell detection reagent according to any one of claims 3 to 7.

9. The test kit according to claim 8, wherein the test kit further comprises an immunofluorescence detection reagent;

Preferably, the immunofluorescence detection reagent is a chromosomal fluorescence in situ hybridization-related reagent;

Preferably, the immunofluorescence detection reagent includes any one or more of the following marker antibodies: tumor marker antibodies, leukocyte marker antibodies, vascular endothelial cell marker antibodies, stem cell marker antibodies, and mesenchymal transition markers Antibodies or endometrial cell marker antibodies;

Preferably, the tumor marker antibody includes an antibody of an epithelial-derived tumor marker; preferably, the epithelial-derived tumor marker is selected from any one or more of the following: estrogen receptor, progesterone receptor, PD-L1, HER2, CEA, CA125, CA19-9, EpCAM, Vimentin, Cytokeratin, Claudin 18.0, Claudin 18.2, AFP, HE4, CD47 or CD74;

Preferably, the leukocyte marker antibody is selected from any one or more of the following: anti-CD3, anti-CD45, anti-CD50, anti-CD69, anti-CD84 or anti-CD102;

Preferably, the vascular endothelial cell marker antibody is selected from any one or more of the following: anti-CD31 or anti-CD146;

Preferably, the stem cell marker antibody is selected from any one or more of the following: anti-CD34, anti-CD44, anti-CD133 or anti-EpCAM;

Preferably, the mesenchymal transition marker antibody is selected from any one or more of the following: vimentin antibody or epithelial cell cadherin antibody.

10. A method for identifying the dead and alive state of cells, characterized in that the method comprises:

The dead cell detection reagent described in any one of claims 3 to 7 is added to the cell sample to be tested for detection.

The method according to claim 10, wherein the cell sample to be tested is a tumor tissue-derived cell sample or a biological fluid-derived cell sample;

Preferably, the tumor tissue-derived cell sample is derived from any one of the following solid tumors: lung cancer, cervical cancer, esophageal cancer, colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, Liver cancer, melanoma, glioma;

Preferably, the biological fluid is selected from any one or more of the following: blood, bone marrow, urine, cerebrospinal fluid, pleural effusion, ascites or lymph node puncture fluid;

Preferably, the cell sample to be tested is a cell suspension.

12. The method according to claim 11, wherein the cell sample to be tested is a sample of various nucleated cells in the biological fluid, including blood-derived tumor cells, non-blood-derived tumor cells, Vascular endothelial cells, stem cells, fetal cells in blood, endometrial cells or blood-derived leukocytes;

Preferably, the blood-borne tumor cells are lymphoma cells;

Preferably, the non-blood-derived tumor cells are solid tumor cells;

Preferably, the stem cells are tumor stem cells.

13. The method according to claim 10, wherein the cell sample to be tested is an immune cell;

Preferably, the immune cells are lymphocytes.

14. A chromosomal fluorescence in situ hybridization method, wherein the method comprises:

Use the dead cell detection reagent according to any one of claims 3 to 7 to stain dead cells in the cell sample to be tested;

Chromosome fluorescence in situ hybridization is performed on the stained cell sample to be tested.

15. The method according to claim 14, wherein, in the dead cell detection reagent, the dead cell fluorescent dye and the fluorescent dye stabilizer are separately stored or placed, and the method comprises:

adding the dead cell dye and an optional fluorescently labeled antibody to the fluorescent dye stabilizer to obtain a mixed solution;

Using the mixture to stain the cell sample to be tested to obtain stained cells;

performing said chromosomal fluorescence in situ hybridization on said stained cells;

Preferably, the fluorescently labeled antibodies include any one or more of the following marker antibodies: tumor marker antibodies, leukocyte marker antibodies, vascular endothelial cell marker antibodies, stem cell marker antibodies, and mesenchymal transition marker antibodies or endometrial cell marker antibodies;

Preferably, the vascular endothelial cell marker antibody is selected from any one or more of the following: anti-CD31, anti-CD146;

Preferably, the stem cell marker antibody is selected from any one or more of the following: anti-CD34, anti-CD44, anti-CD133, and anti-EpCAM;

16. The method according to claim 15, wherein the cell sample to be tested is a tumor tissue-derived cell sample or a biological fluid-derived cell sample;

Preferably, the cell sample to be tested is a cell suspension.

17. The method according to claim 16, wherein the cell sample to be tested is a sample of various nucleated cells in the biological fluid, including blood-derived tumor cells, non-blood-derived tumor cells, Vascular endothelial cells, stem cells, fetal cells in blood, endometrial cells or blood-derived leukocytes;

Preferably, the blood-borne tumor cells are lymphoma cells;

Preferably, the non-blood-derived tumor cells are solid tumor cells;

Preferably, the stem cells are tumor stem cells.

18. The method according to claim 15, wherein the cell sample to be tested is an immune cell;

Preferably, the immune cells are lymphocytes.

19. The method of claim 15, wherein the method comprises:

adding the mixture to the cell suspension of the cell sample to be tested for staining to obtain the stained cells;

Washing, smearing and chromosomal fluorescence in situ hybridization are performed on the stained cells in sequence;

Preferably, the flushing is performed using phosphate buffered saline;

Preferably, the washing is performed by centrifugation at a speed of 1000-1500 rpm for 3-5 minutes;

Preferably, the number of times of the flushing is 2 to 5 times.

20. The method of claim 19, wherein the chromosomal fluorescence in situ hybridization comprises:

The washed stained cells are subjected to gradient dehydration with ethanol to obtain dehydrated cells;

DNA denaturation is performed by placing the dehydrated cells at 70-80° C. to obtain DNA denatured cells;

Using a target fluorescent probe to perform the chromosome fluorescence in situ hybridization on the DNA denatured cells;

Preferably, the target fluorescent probe includes a chromosome probe;

Preferably, the chromosome probes include any one or more of the following: CEP7, CEP8 or ALK fusion gene probes or hematological tumor probes;

Preferably, the hematological tumor probe comprises a lymphoma probe.

21. The fluorescent dye stabilizer according to claim 1 or 2, or the dead cell detection reagent according to any one of claims 3 to 7 or the test kit according to claim 9 or 10, is used to detect live cells and dead cells. The application in the invention is characterized in that the application is a non-diagnostic application.

22. The use of claim 21, wherein the use comprises staining the dead cells with the dead cell detection reagent;

Preferably, on the basis of staining the dead cells, the application further includes: using a method of chromosomal fluorescence in situ hybridization or immunofluorescence antibody staining-chromosomal fluorescence in situ hybridization to discriminate the state of the living cells.