Corn leaf cell nucleus dissociation liquid suitable for analysis of flow cytometry and application of corn leaf cell nucleus dissociation liquid

Abstract

The invention discloses a corn leaf cell nucleus dissociation liquid suitable for flow cytometry analysis and application of the corn leaf cell nucleus dissociation liquid, and belongs to the technical field of flow cytometry analysis. Compared with the traditional liquid nitrogen grinding and digestion technology, the method has the advantages that the corn leaf tissues are infiltrated and treated by using the compounded dissociation liquid, and then the blade scratching operation is performed, so that the problem of large material loss is greatly solved, the sample waste is reduced, the tissue fragment pollution is reduced, the purity and quality of cell nucleuses are improved, and a reliable sample basis is provided for subsequent experiments; compared with a mechanical cutting separation method, dissociation is more sufficient, the requirement for tissue dosage is greatly reduced, the method has great significance to precious and scarce samples, scientific research of a small number of samples is facilitated, and development of related research fields is promoted. The prepared fluorescent probe is free of heavy metal elements, reduces cytotoxicity, is excellent in light stability, small in size, capable of entering a cell nucleus, good in solubility in a complex water environment and suitable for various experiment conditions, and provides powerful support for accurate and stable cell nucleus imaging.

Description

Technical Field

[0001] The invention belongs to the technical field of flow cytometer analysis, and in particular relates to a corn leaf cell nucleus dissociation solution suitable for flow cytometer analysis and application thereof. Background Art

[0002] In the interdisciplinary research field of modern biology and agricultural science, flow cytometry, as a powerful analytical tool, plays a key role in cellular-level research, especially for the analysis of corn leaf nuclei, which can provide indispensable high-precision data and in-depth insights for many scientific research directions such as plant genetics, developmental biology, and crop breeding. If the nuclei of plant tissues are directly isolated and obtained from tissues or organs, such as the nuclei of corn leaf tissues, they have irreplaceable value in scientific research exploration. Its natural state is completely preserved, which means that the nuclei preserve their original genomic information almost intact, providing the most authentic and reliable sample basis for the study of genetic material. At the same time, in actual operation, this direct separation method is relatively simple and efficient, without the need for complex technology and equipment support, so that various scientific research laboratories can carry out related research work more conveniently, which greatly promotes the popularization and in-depth study of corn leaf nuclei.

[0003] At present, common methods for isolating plant cell nuclei include liquid nitrogen grinding, mechanical methods, and protoplast preparation methods. These methods aim to successfully isolate the cell nuclei from leaf tissues so that they can be directly applied to subsequent scientific research, such as chromatin accessibility analysis, chromatin immunoprecipitation, and other transformation experiments. Directly isolated corn leaf tissue cell nuclei have significant advantages. Their in vitro time is short, which effectively avoids changes in the physiological state of cells and loss of genetic information that may be caused by long-term in vitro isolation. Moreover, since they have not undergone the immortalization process, their biological characteristics are highly consistent with their actual state in the body, and their genetic characteristics are stable and reliable, thus providing researchers with data that is closest to the actual physiological function of the cell nucleus in the body, which is of great significance for in-depth understanding of the life activities and genetic regulation mechanisms of corn leaf cells.

[0004] However, when using flow cytometry to analyze the nuclei of corn leaves, the key step of nucleus staining faces many technical difficulties. Since the nucleus is tightly wrapped by a special lipid bilayer membrane, the fluorescent probe must penetrate this barrier to effectively stain the nucleus and meet the detection requirements of the flow cytometer. In theory, the size of the fluorescent probe should be smaller than the 8nm nuclear pore to have the possibility of entering the nucleus, but in fact, it is far from enough to meet this size condition, because whether the probe can successfully enter the nucleus is also significantly affected by many factors such as its surface properties.

[0005] In traditional nuclear staining studies, organic dyes such as 4',6-diamidino-2-phenylindole and Hoechst dyes have been widely used in nuclear staining of cancer cells and normal cells because of their certain biocompatibility. However, these organic dyes have insurmountable defects. They have poor water solubility and are prone to agglomeration in aqueous environments, which makes it difficult to ensure the uniformity of staining, thereby affecting the accuracy and reliability of flow cytometer detection. In addition, their photostability is insufficient. During flow cytometer detection, especially under long-term laser irradiation, the fluorescence intensity will drop rapidly, that is, photobleaching occurs, which makes the detection signal unstable and cannot meet the needs of high-precision, long-term detection and analysis of cell nuclei, which seriously limits its further application in flow cytometer analysis of corn leaf cell nuclei. On the other hand, semiconductor quantum dots such as cadmium telluride quantum dots and cadmium selenide quantum dots have also shown certain application potential in the field of cell fluorescence imaging. However, this type of quantum dots has serious safety risks. They contain heavy metal elements, such as cadmium, which are highly toxic and may cause a series of adverse physiological reactions in the body. They will not only interfere with the normal metabolism and function of cells, but also threaten the health of the entire organism. This makes them face strict safety restrictions in biological imaging, especially flow cytometer analysis. Moreover, if semiconductor quantum dots are not modified in a complex way, their efficiency in penetrating the nuclear membrane and entering the nucleus is low, making it difficult to achieve efficient staining and accurate analysis of the nucleus, and unable to meet the requirements of flow cytometry for high-quality staining of nuclear samples. Summary of the invention

[0006] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides an efficient and simple dissociation solution and dissociation method for corn leaf cell nuclei, which can be used for flow cytometric detection of corn leaf cell nuclei.

[0007] In order to achieve the above object, the following technical scheme is adopted: The present invention provides an application of a corn leaf cell nucleus dissociation solution suitable for flow cytometer analysis, comprising the following steps:

[0008] a. Collect fresh corn leaves with 2 true leaves, place the leaf tissue on a pre-cooled glass plate, add 1 mL of pre-cooled dissociation solution, and quickly scratch with a sterile surgical blade for 2 minutes. After mincing, rinse the minced sample with 4 mL of pre-cooled dissociation solution into a sterile 15 mL EP centrifuge tube, place on a shaker at room temperature for 5 minutes to dissociate the corn leaf tissue, and obtain a corn leaf tissue cell nucleus solution;

[0009] b. centrifuging the corn leaf tissue cell nucleus solution at low speed in a centrifuge precooled at 4°C, removing the supernatant, and obtaining the corn leaf tissue cell nucleus precipitate;

[0010] c. Add 5 mL of dissociation washing solution to the corn leaf tissue cell nuclear precipitate to resuspend it, centrifuge it at a low speed in a 4°C precooled centrifuge, remove the supernatant, and obtain a purified corn leaf tissue cell nuclear precipitate;

[0011] d. Add 1 mL of dissociation washing solution to the purified corn leaf tissue cell nuclear precipitate and resuspend it. After mixing, use a pipette to draw 10 μL of the upper cell solution into a new EP tube, then add an equal volume of carbon quantum dot fluorescent probe for staining, and observe the integrity of the cell nucleus under a microscope;

[0012] e. Collect cell nuclei from corn leaf tissue and perform flow cytometric analysis;

[0013] Furthermore, the carbon quantum dot fluorescent probe is prepared by the following steps:

[0014] S1. Add 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine to a mixed solution of ethylene glycol and DMF, and then ultrasonicate for 10-15 minutes to obtain a first solution. Then, add the first solution to a polytetrafluoroethylene hydrothermal reactor, add a 0.5 mol / L DMF solution of perfluorohexyl iodide and an organic metal initiator, and perform a hydrothermal reaction at 180-220° C. for 3-15 hours;

[0015] S2, after the reaction is completed, the hydrothermal reactor is cooled to room temperature to obtain a second solution, which is filtered and dialyzed for purification;

[0016] S3, removing the solvent from the dialyzed and purified solution under reduced pressure, and obtaining the carbon quantum dot fluorescent probe after drying.

[0017] Furthermore, in the first solution of step S1, the mass ratio of 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine, ethylene glycol and DMF is 0.5-2:10-20:20-50.

[0018] Furthermore, in step S1, the mass ratio of the first solution, the DMF solution of perfluorohexyl iodide and the organic metal initiator is 10-30: 5-10: 0.01-0.05.

[0019] Furthermore, the organic metal initiator is selected from one of acetylferrocene, tris(acetylacetonate)ferrocene, diethylzinc and zirconocene dichloride.

[0020] Furthermore, the filtration in step S2 uses a 0.2 μm or 0.22 μm aqueous filter membrane.

[0021] Furthermore, the dialysis in step S2 is performed using a dialysis bag with a cutoff of 1 kD for 12-18 hours.

[0022] The present invention also provides a corn leaf cell nucleus dissociation solution suitable for flow cytometer analysis, wherein the dissociation solution consists of the following components: 4 mol / L MES-KOH solution with a pH value of 5.7, 5 mol / L NaCl solution, 1 mol / L KCl solution, 1 mol / L MgCl2 solution, 0.5 mol / L EDTA solution and 4 mol / L sucrose solution.

[0023] Furthermore, the volume ratio of the MES-KOH solution, the NaCl solution, the KCl solution, the MgCl2 solution, the EDTA solution and the sucrose solution in the dissociation solution is 5:4:20:6:10:125.

[0024] Furthermore, the dissociation cleaning solution consists of the following components: 4 mol / L MES-KOH solution with a pH of 5.7, 5 mol / L NaCl solution, 1 mol / L KCl solution, 1 mol / L MgCl2 solution and 4 mol / L sucrose solution.

[0025] Furthermore, the volume ratio of the MES-KOH solution, the NaCl solution, the KCl solution, the MgCl2 solution and the sucrose solution in the dissociation cleaning solution is 5:4:20:6:125.

[0026] The beneficial effects of the present invention are:

[0027] a. The present invention develops a new method for dissociating corn leaf nuclei. After the corn leaf tissue is infiltrated with a compound corn leaf nucleus dissociation solution, a blade scratch operation method is used to achieve the dissociation process, so that the corn leaf tissue nuclei can be quickly obtained in a short time. Compared with the traditional liquid nitrogen grinding and digestion technology, the present invention effectively solves the problem of excessive material loss, greatly reduces the waste of samples, and significantly reduces the contamination degree of tissue fragments, improves the purity and quality of the obtained nuclei, and provides a more reliable sample basis for subsequent experimental research. In addition, compared with the mechanical cutting and separation method, the dissociation effect of the present invention is more sufficient, and the requirement for the amount of tissue is greatly reduced, and a tissue amount greater than 1g is no longer required, which is of great significance for precious and scarce samples, greatly saves sample resources, and enables scientific research work to be carried out smoothly with a small amount of samples, providing strong support for the further expansion of related research fields;

[0028] b. The present invention uses 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine as a key starting material to prepare carbon quantum dots. During the preparation process, an organic metal initiator is introduced to initiate perfluoroiodohexane to decompose fluorine to produce fluorine free radicals or fluorine ions, thereby fluorine doping the carbon quantum dots. The carbon quantum dot fluorescent probe prepared by the present invention has a special structure of nitrogen and fluorine dual doping. First, it does not contain heavy metal elements, which reduces the toxicity to cells, so that in applications such as cell imaging, it can minimize the interference with the physiological state of cells and ensure the accuracy and reliability of experimental results; secondly, it has excellent light stability, and can still maintain stable fluorescence performance under long-term illumination conditions, providing a solid guarantee for long-term, high-precision nuclear imaging; furthermore, it is small in size and can smoothly pass through the membrane structure of the cell nucleus, meeting the requirements for entering the cell nucleus for staining and imaging; in addition, the fluorescent probe still shows good solubility in a complex water environment with a variety of metal ions, ensuring its stability and reliability in practical applications, and can adapt to various complex experimental conditions, providing reliable support for accurate and stable nuclear imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a graph showing the particle size analysis results of the carbon quantum dot fluorescent probe of Example 3 of the present invention;

[0030] Figure 2 This is a diagram showing the cytotoxicity analysis results of the carbon quantum dot fluorescent probe of Example 1 of the present invention;

[0031] Figure 3 This is a diagram showing the cytotoxicity analysis results of the carbon quantum dot fluorescent probe of Example 2 of the present invention;

[0032] Figure 4 This is a diagram showing the cytotoxicity analysis results of the carbon quantum dot fluorescent probe of Example 3 of the present invention;

[0033] Figure 5 This is a fluorescence staining result diagram of the corn leaf tissue after obtaining the cell nucleus of the present invention;

[0034] Figure 6 This is a flow cytometer analysis result diagram of obtaining cell nuclei from corn leaf tissue of the present invention.

[0035] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0037] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0039] Example 1

[0040] Composition of maize leaf cell nucleus dissociation solution and dissociation washing solution suitable for flow cytometric analysis

[0041] The dissociation solution consists of the following components: 4 mol / L MES-KOH solution with pH=5.7, 5 mol / L NaCl solution, 1 mol / L KCl solution, 1 mol / L MgCl2 solution, 0.5 mol / L EDTA solution and 4 mol / L sucrose solution.

[0042] The volume ratio of the MES-KOH solution, the NaCl solution, the KCl solution, the MgCl2 solution, the EDTA solution and the sucrose solution in the dissociation solution is 5:4:20:6:10:125.

[0043] The dissociation cleaning solution consists of the following components: 4 mol / L MES-KOH solution with a pH of 5.7, 5 mol / L NaCl solution, 1 mol / L KCl solution, 1 mol / L MgCl2 solution and 4 mol / L sucrose solution.

[0044] The volume ratio of the MES-KOH solution, the NaCl solution, the KCl solution, the MgCl2 solution and the sucrose solution in the dissociation cleaning solution is 5:4:20:6:125.

[0045] Example 2

[0046] Composition and preparation of carbon quantum dot fluorescent probe

[0047] The carbon quantum dot fluorescent probe is prepared by the following steps:

[0048] S1. Add 2g of 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine to a mixed solution of 20g of ethylene glycol and 50g of DMF, and perform ultrasonic treatment for 15min to obtain a first solution. Then add the first solution to a polytetrafluoroethylene hydrothermal reactor, add 10g of a DMF solution of perfluorohexane with a concentration of 0.5mol / L and 0.05g of zirconocene dichloride as an organic metal initiator, and perform a hydrothermal reaction at 220°C for 15h.

[0049] S2. After the reaction is completed, the hydrothermal reactor is cooled to room temperature, filtered with a 0.22 μm aqueous filter membrane, and then dialyzed for 18 hours using a dialysis bag with a cutoff of 1 kD for purification.

[0050] S3, removing the solvent from the dialyzed and purified solution under reduced pressure, and obtaining the carbon quantum dot fluorescent probe after drying.

[0051] Example 3

[0052] Composition and preparation of carbon quantum dot fluorescent probe

[0053] The carbon quantum dot fluorescent probe is prepared by the following steps:

[0054] S1. Add 0.5 g of 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine to a mixed solution of 10 g of ethylene glycol and 20 g of DMF, and perform ultrasonic treatment for 10 min to obtain a first solution. Then add the first solution to a polytetrafluoroethylene hydrothermal reactor, add 5 g of a DMF solution of 0.5 mol / L perfluorohexane iodide and 0.01 g of acetylferrocene as an organic metal initiator, and perform a hydrothermal reaction at 180°C for 3 h.

[0055] S2. After the reaction is completed, the hydrothermal reactor is cooled to room temperature, filtered with a 0.2 μm aqueous filter membrane, and then dialyzed for 12 hours using a dialysis bag with a retention capacity of 1 kD for purification.

[0056] S3, removing the solvent from the dialyzed and purified solution under reduced pressure, and obtaining the carbon quantum dot fluorescent probe after drying.

[0057] Example 4

[0058] Composition and preparation of carbon quantum dot fluorescent probe

[0059] The carbon quantum dot fluorescent probe is prepared by the following steps:

[0060] S1. Add 1 g of 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine to a mixed solution of 15 g of ethylene glycol and 30 g of DMF, and perform ultrasonic treatment for 12 min to obtain a first solution. Then add the first solution to a polytetrafluoroethylene hydrothermal reactor, add 7 g of a DMF solution of perfluorohexane with a concentration of 0.5 mol / L and 0.03 g of tris(acetylacetonate)iron as an organic metal initiator, and perform a hydrothermal reaction at 200°C for 8 h.

[0061] S2. After the reaction is completed, the hydrothermal reactor is cooled to room temperature, filtered with a 0.2 μm aqueous filter membrane, and then dialyzed for 15 h using a dialysis bag with a retention capacity of 1 kD for purification.

[0062] S3, removing the solvent from the dialyzed and purified solution under reduced pressure, and obtaining the carbon quantum dot fluorescent probe after drying.

[0063] Test Example 1

[0064] Carbon quantum dot fluorescent probe particle size analysis

[0065] The average particle size distribution of Example 4 was measured by a dynamic light scattering particle size analyzer. Figure 1 .

[0066] Depend on Figure 2 It can be seen that the particle size of the carbon quantum dot fluorescent probe prepared in Example 3 is less than 8nm, which can meet the conditions for entering the cell nucleus. This result directly reflects the advantages of the carbon quantum dot fluorescent probe preparation scheme of the present invention. The present invention successfully prepares a probe with a suitable particle size by precisely controlling the raw material ratio, reaction conditions, etc. The smaller particle size enables it to penetrate the lipid bilayer membrane of the cell nucleus, overcoming the obstacle that traditional fluorescent probes are difficult to enter the cell nucleus due to particle size problems, and provides the possibility for subsequent precise staining and analysis of the cell nucleus, ensuring that more accurate and detailed information inside the cell nucleus can be obtained, which is of great significance for in-depth research on the structure and function of the cell nucleus. For example, when studying the distribution and state of chromatin, it can more clearly present its true situation in the cell nucleus, avoiding information loss caused by the inability of the probe to enter.

[0067] Test Example 2

[0068] Analysis of cytotoxicity using carbon quantum dot fluorescent probes

[0069] The carbon quantum dot fluorescent probes of Examples 2-4 were prepared into solutions with concentrations of 0, 1000, 500, 250, 125, 75, 40, 20, 10, 5, and 2 μg / mL, and cell activity was detected. The results are shown in Figure 2-4 .

[0070] Depend on Figure 2-4It does not contain heavy metal elements and has low cytotoxicity. The carbon quantum dot fluorescent probe of the present invention uses an organic metal initiator to achieve fluorine doping during the preparation process, avoiding the use of heavy metals in traditional semiconductor quantum dots and significantly reducing toxicity to cells. In applications such as cell imaging, low cytotoxicity ensures that cells can maintain normal physiological states and functional activities, so that the experimental results can truly reflect the characteristics and behaviors of the cell nucleus in a natural state. In long-term cell culture observation experiments, cells will not show abnormal metabolic changes, abnormal gene expression or cell death due to the toxicity of the probe. This provides a reliable guarantee for accurately studying the changing laws of the cell nucleus during the cell cycle, cell differentiation and other processes, and improves the credibility of experimental data and the accuracy of scientific research conclusions.

[0071] Test Example 3

[0072] The cell nuclei of corn leaf tissue were extracted using the cell nucleus dissociation solution and the dissociation cleaning solution in Example 1, and the cell nuclei were stained and observed using the carbon quantum dot fluorescent probe in Example 4, specifically comprising the following steps:

[0073] a. Collect fresh corn leaves with 2 true leaves, place the leaf tissue on a pre-cooled glass plate, add 1 mL of pre-cooled dissociation solution, and quickly scratch with a sterile surgical blade for 2 minutes. After mincing, rinse the minced sample with 4 mL of pre-cooled dissociation solution into a sterile 15 mL EP centrifuge tube, place on a shaker at room temperature for 5 minutes to dissociate the corn leaf tissue, and obtain a corn leaf tissue cell nucleus solution;

[0074] b. centrifuging the corn leaf tissue cell nucleus solution at low speed in a centrifuge precooled at 4°C, removing the supernatant, and obtaining the corn leaf tissue cell nucleus precipitate;

[0075] c. Add 5 mL of dissociation washing solution to the corn leaf tissue cell nuclear precipitate to resuspend it, centrifuge it at a low speed in a 4°C precooled centrifuge, remove the supernatant, and obtain a purified corn leaf tissue cell nuclear precipitate;

[0076] d. Add 1 mL of dissociation washing solution to the purified corn leaf tissue cell nuclear precipitate and resuspend it. After mixing, use a pipette to draw 10 μL of the upper cell solution into a new EP tube, then add an equal volume of carbon quantum dot fluorescent probe for staining, and observe the integrity of the cell nuclear under a microscope. The results are shown in Figure 5 .

[0077] Depend on Figure 5It can be seen that the results show that the extracted cell nuclei are relatively complete, and the tissue fragments are almost completely removed, which can meet the needs of subsequent scientific research. The dissociation solution and unique blade scratch dissociation method used in the present invention are a new and efficient cell nucleus extraction scheme compared to traditional liquid nitrogen grinding and digestion techniques. On the one hand, it effectively solves the problem of excessive material loss and greatly reduces the waste of samples. For precious and scarce corn sample resources, a small amount of samples can meet the experimental needs, which has great advantages when germplasm resources are limited or specific genetic research materials are rare; on the other hand, it significantly reduces the degree of contamination of tissue fragments, improves the purity and quality of the obtained cell nuclei, and provides a more reliable sample basis for subsequent experiments. At the same time, compared with the mechanical cutting and separation method, the dissociation effect is more sufficient, the requirement for tissue dosage is greatly reduced, and it no longer relies on a large amount of tissue materials, making the experimental operation simpler, more flexible, and reducing costs. Combined with the carbon quantum dot fluorescent probe of the present invention, the complete cell nucleus can be effectively stained, further ensuring the accurate morphological observation and analysis of the cell nucleus, providing clear and accurate sample images for studying the morphological structure, chromosome number and other basic characteristics of the corn leaf cell nucleus, and facilitating in-depth research in fields such as cell genetics.

[0078] Test Example 4

[0079] The cell nuclei of corn leaf tissue were collected and analyzed by flow cytometry. The results are shown in Figure 6 .

[0080] Depend on Figure 6 It can be seen that the fluorescence value of the corn leaf tissue nucleus sample is 127805, and its CV value is 3.84%, which is calculated to be diploid, consistent with the literature value. The above results fully demonstrate the effectiveness and reliability of the combination of the cell nucleus dissociation solution, dissociation cleaning solution and carbon quantum dot fluorescent probe of the present invention in flow cytometer analysis. The high-quality cell nucleus dissociation and staining effect provides a stable and uniform fluorescence signal for the flow cytometer, so that the instrument can accurately detect and analyze various parameters of the cell nucleus, such as cell cycle distribution, DNA content, etc. Compared with traditional cell nucleus processing and staining methods, the technical solution of the present invention can better meet the strict requirements of flow cytometers for sample quality and improve the accuracy and repeatability of the test results. In plant cell biology research, this is of great value for in-depth understanding of the proliferation, differentiation, genetic stability and other aspects of corn leaf cells.

[0081] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0082] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. An application of a corn leaf nucleus dissociation solution suitable for flow cytometer analysis, characterized in that: The following steps are involved: a. Collect fresh corn leaves with 2 true leaves, place the leaf tissue on a pre-cooled glass plate, add 1 mL of pre-cooled dissociation solution, and quickly scratch with a sterile surgical blade for 2 minutes. After mincing, rinse the minced sample with 4 mL of pre-cooled dissociation solution into a sterile 15 mL EP centrifuge tube, place on a shaker at room temperature for 5 minutes to dissociate the corn leaf tissue, and obtain a corn leaf tissue cell nucleus solution; b. centrifuging the corn leaf tissue cell nucleus solution at low speed in a centrifuge precooled at 4°C, removing the supernatant, and obtaining the corn leaf tissue cell nucleus precipitate; c. Add 5 mL of dissociation washing solution to the corn leaf tissue cell nuclear precipitate to resuspend it, centrifuge it at a low speed in a 4°C precooled centrifuge, remove the supernatant, and obtain a purified corn leaf tissue cell nuclear precipitate; d. Add 1 mL of dissociation washing solution to the purified corn leaf tissue cell nuclear precipitate and resuspend it. After mixing, use a pipette to draw 10 μL of the upper cell solution into a new EP tube, then add an equal volume of carbon quantum dot fluorescent probe for staining, and observe the integrity of the cell nucleus under a microscope; e. Collect cell nuclei from corn leaf tissue and perform flow cytometric analysis; The carbon quantum dot fluorescent probe is prepared by the following steps: S1. Add 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine to a mixed solution of ethylene glycol and DMF, and then ultrasonicate for 10-15 minutes to obtain a first solution. Then, add the first solution to a polytetrafluoroethylene hydrothermal reactor, add a 0.5 mol / L DMF solution of perfluorohexyl iodide and an organic metal initiator, and perform a hydrothermal reaction at 180-220° C. for 3-15 hours; S2, after the reaction is completed, the hydrothermal reactor is cooled to room temperature to obtain a second solution, which is filtered and dialyzed for purification; S3, removing the solvent from the dialyzed and purified solution under reduced pressure, and obtaining the carbon quantum dot fluorescent probe after drying.

2. The use of the corn leaf nucleus dissociation solution suitable for flow cytometer analysis according to claim 1, characterized in that: The mass ratio of 3,3',6,6'-tetracarboxylic acid-2,2'-bipyridine, ethylene glycol and DMF in the first solution of step S1 is 0.5-2:10-20:20-50.

3. The use of the corn leaf nucleus dissociation solution suitable for flow cytometer analysis according to claim 2, characterized in that: In the step S1, the mass ratio of the first solution, the DMF solution of perfluorohexyl iodide and the organic metal initiator is 10-30: 5-10: 0.01-0.

05.

4. The use of the corn leaf nucleus dissociation solution suitable for flow cytometer analysis according to claim 3, characterized in that: The organic metal initiator is selected from one of acetylferrocene, tris(acetylacetonate)ferrocene, diethylzinc and zirconocene dichloride.

5. The use of the corn leaf nucleus dissociation solution suitable for flow cytometer analysis according to claim 4, characterized in that: The filtration in step S2 uses a 0.2 μm or 0.22 μm aqueous filter membrane.

6. The use of the corn leaf nucleus dissociation solution suitable for flow cytometer analysis according to claim 5, characterized in that: In step S2, the dialysis is performed using a dialysis bag with a cutoff of 1 kD for 12-18 hours.

7. A corn leaf nucleus dissociation solution suitable for flow cytometric analysis according to any one of claims 1 to 6, characterized in that: The dissociation solution consists of the following components: 4 mol / L MES-KOH solution with pH=5.7, 5 mol / L NaCl solution, 1 mol / L KCl solution, 1 mol / L MgCl2 solution, 0.5 mol / L EDTA solution and 4 mol / L sucrose solution.

8. The corn leaf nucleus dissociation solution suitable for flow cytometer analysis according to claim 7, characterized in that: The volume ratio of the MES-KOH solution, the NaCl solution, the KCl solution, the MgCl2 solution, the EDTA solution and the sucrose solution in the dissociation solution is 5:4:20:6:10:

125.

9. The corn leaf nucleus dissociation solution suitable for flow cytometer analysis according to claim 8, characterized in that: The dissociation cleaning solution consists of the following components: 4 mol / L MES-KOH solution with a pH of 5.7, 5 mol / L NaCl solution, 1 mol / L KCl solution, 1 mol / L MgCl2 solution and 4 mol / L sucrose solution.

10. The corn leaf cell nucleus dissociation solution suitable for flow cytometer analysis according to claim 9, characterized in that: The volume ratio of the MES-KOH solution, the NaCl solution, the KCl solution, the MgCl2 solution and the sucrose solution in the dissociation cleaning solution is 5:4:20:6:125.

Images