Application of CrCl2 in detecting cell apoptosis using TUNEL technique

Abstract

The present invention discloses the application of CrCl2 in the TUNEL technique for detecting cell apoptosis. The present invention first discloses the application of CrCl2 in the TUNEL technique for detecting cell apoptosis. Further disclosed is a TdT reaction buffer system, which comprises HEPES, NaCl, CoCl2, MgCl2, DTT, dATP and CrCl2. The present invention first discovered that the presence of CrCl2 in the TdT reaction buffer system can catalyze the TdT reaction and provide a better reaction environment for the terminal deoxynucleotidyl transferase reaction. The system is applicable to both TUNEL kits based on Click reaction and traditional TUNEL kits. The TdT reaction buffer system containing CrCl2 has low toxicity and has the advantages of high detection sensitivity, strong specificity and simple and rapid operation steps when detecting cell apoptosis using the TUNEL technique. The system can be used for the detection of different cell and tissue samples.

Description

Technical Field

[0001] The present invention relates to the field of cell detection technology, and more specifically, to the application of CrCl2 in detecting cell apoptosis using TUNEL technology. Background Art

[0002] Apoptosis, also known as programmed cell death, is crucial for the evolution of organisms, the stability of their internal environment, and the development of multiple systems. Apoptosis is not only a specific type of cell death but also has important biological significance and complex molecular biological mechanisms (Kerr, J, FR, AH Wyllie, and A.R. Ctlrrie. 1972. Apoptosis: basic biological henomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer. 26:239-257.; Wyllie AH: Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 1980, 284:555-556). However, the exact mechanisms of apoptosis remain largely unknown. Disruptions in the apoptotic process may be directly or indirectly linked to the development of many diseases, such as tumors and autoimmune diseases, and components of the apoptotic pathway could serve as therapeutic targets. Therefore, the ability to identify and quantify apoptosis is crucial for advancing research in these areas.

[0003] The TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end labeling (TUNEL) technique was first published in late 1992. TUNEL biochemically labels exposed DNA ends, allowing visualization of cell nuclei containing DNA fragments. One of the biochemical hallmarks of apoptosis is the fragmentation of chromatin into single or multiple oligonucleotide-length fragments, resulting in the generation of free 3'-hydroxyl termini on the DNA. TUNEL staining utilizes terminal deoxynucleotidyl transferase to bind labeled dUTP to free 3'-hydroxyl termini on genomic DNA fragments. Although TUNEL is used as a method for detecting apoptosis in situ, it is not limited to detecting apoptotic cells (Watanabe M, Hitomi M, van der Wee K, Rothenberg F, Fisher SA, Zucker R, Svoboda KK, Goldsmith EC, Heiskanen KM, Nieminen AL. The pros and cons of apoptosis assays for use in the study of cells, tissues, and organs. Microsc Microanal 2002; 8: 375–91.). In a sense, TUNEL is nonspecific. It does not take into account the formation mechanism of these DNA ends and thus labels all free 3'-hydroxyl termini. It can also detect non-apoptotic cells, including necrotic cells, DNA repair cells, physically damaged cells and even cells with active gene transcription (Vladimir V. Didenko (ed.), DNA Damage Detection In Situ, Ex Vivo, and In Vivo: Methods and Protocols, Methods in Molecular Biology, vol. 682, DOI 10.1007 / 978-1-60327-409-8_2).

[0004] Currently, the TUNEL cell apoptosis detection kits on the market mainly include traditional TUNEL kits and TUNEL kits based on Click reaction. Traditional TUNEL kits mainly use antibodies or dUTP macromolecules modified with fluorescein or biotin. Their molecular weight is large, not easily incorporated by TdT enzyme, and the product fluorescence is single. TUNEL kits based on Click reaction use dUTP modified with alkyne groups (EdUTP), which is not only easily incorporated by TdT enzyme, but also detects a higher proportion of apoptotic cells under the same conditions, and has multi-color fluorescence options, but the two-step method has a longer experimental cycle than the one-step method. At this stage, the technology for TUNEL detection of cell apoptosis is constantly innovating, but cell biology experiments basically face problems including strong non-specificity, strong background, and low staining efficiency.

[0005] Therefore, the present invention has developed a new buffer reaction solution system for TUNEL technology to solve the above problems. Summary of the Invention

[0006] The first object of the present invention is to provide the application of CrCl2 in detecting cell apoptosis using TUNEL technology.

[0007] The second object of the present invention is to provide a terminal deoxynucleotidyl transferase (TdT) reaction buffer system with low toxicity, which can provide a better reaction environment for TdT reaction when detecting cell apoptosis using TUNEL technology, thereby improving sensitivity and reducing nonspecificity.

[0008] The third object of the present invention is to provide an application of a TdT reaction buffer system in detecting cell apoptosis using TUNEL technology.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] According to the first object of the present invention, the present invention provides the use of CrCl2 in detecting cell apoptosis using the TUNEL technique or in preparing a product for detecting cell apoptosis using the TUNEL technique.

[0011] Optionally, the concentration of CrCl2 is 0.01-0.5mM; preferably, it is 0.1mM.

[0012] According to the second object of the present invention, the present invention provides a TdT reaction buffer system, which comprises HEPES, NaCl, CoCl2, MgCl2, DTT, dATP and CrCl2; preferably, the pH of the HEPES is 7.2.

[0013] Optionally, the concentration of CrCl2 is 0.01-0.5mM; preferably, it is 0.1mM.

[0014] Optionally, the concentration of HEPES is 10-60 mmol / L, the concentration of NaCl is 100-150 mM, the concentration of CoCl2 is 1-10 mM, the concentration of MgCl2 is 0.1-1 mM, the concentration of DTT is 2-10 mM, and the concentration of dATP is 1-20 mM.

[0015] A kit comprising the above-mentioned TdT reaction buffer system is also within the scope of protection of the present invention.

[0016] According to the third object of the present invention, the present invention provides the use of the above-mentioned TdT reaction buffer system in detecting cell apoptosis using the TUNEL technique, or in preparing a product for detecting cell apoptosis using the TUNEL technique.

[0017] The TUNEL technique described in the present invention includes both the TUNEL technique based on Click reaction and the traditional TUNEL technique (using dUTP labeled with fluorescein, biotin or antibody).

[0018] The beneficial effects of the present invention are as follows:

[0019] This study first discovered that the presence of CrCl2 in a TdT reaction buffer system can catalyze the TdT reaction, providing an optimal reaction environment for the terminal deoxynucleotidyl transferase (TdT) reaction and enhancing TdT activity. This finding is applicable to both Click-based TUNEL kits and traditional TUNEL kits. The TdT reaction buffer system containing CrCl2 offers the following advantages when using the TUNEL technique to detect apoptosis:

[0020] 1. High detection sensitivity, which can amplify lower apoptosis signals and detect a higher proportion of apoptotic cells;

[0021] 2. Strong specificity, high staining efficiency, clean fluorescence background, greatly reducing the non-specificity of cytoplasmic staining;

[0022] 3. Extremely low toxicity, does not contain the highly toxic organic arsenic compound buffer system that the traditional TUNEL reaction relies on;

[0023] 4. The operation steps are simple and fast;

[0024] 5. Can be used for the detection of different cell and tissue samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0026] Figure 1 Schematic diagram of the experimental principle of TUNEL technology for detecting cell apoptosis.

[0027] Figure 2 This is a diagram showing the staining effect of CrCl2 at different concentration gradients on TUNEL technology for detecting cell apoptosis.

[0028] Figure 3 These are staining results of a mainstream competitor and a mainstream competitor using the TdT reaction buffer system of the present invention to detect cell apoptosis. The unit of the icon is 100 μm.

[0029] Figure 4 This is a diagram showing the staining effect of a traditional competitor and a traditional competitor using the TdT reaction buffer system of the present invention when detecting cell apoptosis using the traditional TUNEL technique.

[0030] Figure 5 The staining effect diagram of detecting cell apoptosis using TdT reaction buffer system with different combinations of TUNEL technology. DETAILED DESCRIPTION

[0031] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the following detailed description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0032] Unless otherwise specified, the test methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] The following examples use the Click-iT technology based on the TUNEL technique to detect apoptosis. TM TUNEL Alexa Fluor TM 488 Imaging Assay, for microscopy & HCS products (hereinafter referred to as “mainstream competitor products”) were purchased from Thermo Fisher Scientific.

[0035] The conventional TUNEL technique used in the following examples was used to detect cell apoptosis. In Situ Fluorescein TUNEL Cell Apoptosis Detection Kit (hereinafter referred to as "traditional competitor") was purchased from TransGen.

[0036] The principle of TUNEL technique for detecting cell apoptosis in the following examples is as follows: Figure 1Specifically, after apoptotic cells are fixed and permeabilized (tissue sections are dewaxed, rehydrated, and permeabilized), the sticky 3'-OH ends of the broken double-stranded or single-stranded genomic DNA are exposed. Under the catalysis of terminal deoxynucleotidyl transferase (TdT), EdUTP / fluorescein-dUTP binds to the 3'-OH ends, thereby labeling the broken DNA ends and amplifying the apoptosis signal.

[0037] In the following example, the steps for detecting cell apoptosis using the TUNEL technique for alkyne-labeled dUTP (detecting cell apoptosis using the TUNEL technique based on the Click reaction) are as follows:

[0038] The cell samples were fixed and permeabilized, and then subjected to TdT reaction;

[0039] TdT reaction: The TdT reaction solution was prepared according to Table 1. The entire TdT reaction was incubated in a 37°C incubator for 60 minutes, washed three times with 3% BSA, and then the Click reaction was performed;

[0040] Table 1 TdT reaction solution

[0041] Components volume TdT reaction buffer system 95μL EdUTP 2μL TdT 3μL Total volume 100 μL

[0042] Click reaction: The Click reaction solution was prepared as shown in Table 2. The entire Click reaction was carried out in a light-proof environment, incubated at room temperature for 30 minutes, and washed three times with PBS. The cell nucleus was labeled with the nuclear dye Hoechst, and then fluorescence in situ microscopy was performed.

[0043] Table 2 Click reaction solution

[0044] Components volume Click reaction buffer system 92.5μL EBA 2.5 μL Total volume 100 μL

[0045] Example 1 TdT reaction buffer system for detecting cell apoptosis using TUNEL technique

[0046] A TdT reaction buffer system (combination 1) for detecting cell apoptosis using the TUNEL technique is composed of the following components:

[0047] 20mmol / L HEPES (pH 7.2), 100mM NaCl, 1mM CoCl2, 0.1mM MgCl2, 2mM DTT, 1mMdATP and 0.01mM CrCl2.

[0048] Example 2 TdT reaction buffer system for detecting cell apoptosis using TUNEL technique

[0049] A TdT reaction buffer system (combination 2) for detecting cell apoptosis using the TUNEL technique is composed of the following components:

[0050] 20mmol / L HEPES (pH 7.2), 137mM NaCl, 5mM CoCl2, 0.5mM MgCl2, 6mM DTT, 10mMdATP and 0.2mM CrCl2.

[0051] Example 3 TdT reaction buffer system for detecting cell apoptosis using TUNEL technique

[0052] A TdT reaction buffer system (combination 3) for detecting cell apoptosis using the TUNEL technique is composed of the following components:

[0053] 50mmol / L HEPES (pH 7.2), 150mM NaCl, 10mM CoCl2, 1mM MgCl2, 10mM DTT, 20mMdATP and 0.5mM CrCl2.

[0054] Example 4 TdT reaction buffer system for detecting cell apoptosis using TUNEL technique

[0055] A TdT reaction buffer system for detecting cell apoptosis using the TUNEL technique, comprising the following components:

[0056] 20mmol / L HEPES (pH 7.2), 137mM NaCl, 5mM CoCl2, 0.5mM MgCl2, 6mM DTT, 10mMdATP and 0.1mM CrCl2.

[0057] Experimental Example 1 Effect of CrCl2 on TUNEL-based apoptosis detection

[0058] The most critical step affecting TUNEL detection of apoptosis in this invention is the TdT reaction. The TdT reaction buffer system is used to maintain specific reaction conditions. A good reaction buffer system should not only enhance enzyme activity and catalyze the reaction, but also meet the requirements of low background and high specificity for in situ detection. The inventors have discovered that CrCl2 has a certain effect on TUNEL detection of apoptosis, as follows:

[0059] On the premise of determining the concentrations of other components (i.e., 20mmol / L HEPES (pH 7.2), 137mM NaCl, 5mM CoCl2, 0.5mM MgCl2, 6mM DTT and 10mM dATP), a CrCl2 concentration gradient was set (the concentrations of CrCl2 were 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.5mM and 1mM, respectively) to prepare TdT reaction buffer systems with different CrCl2 concentrations. On the premise of ensuring that the other components of the mainstream competitor were consistent, only the TdT reaction buffer system in the mainstream competitor was changed to the TdT reaction buffer system with different CrCl2 concentrations mentioned above. According to the "Steps for detecting cell apoptosis using TUNEL technology for alkyne-labeled dUTP", the apoptosis of HeLa cells was detected to determine the optimal CrCl2 concentration, and the cell nuclei were stained with Hoechst. The results are as follows: Figure 2 As shown in the figure, the final concentration of CrCl2 was finally determined to be 0.1mM, thereby finally determining the complete TdT reaction buffer system, that is, the optimal TdT reaction buffer system for TUNEL technology detection of cell apoptosis was 20mmol / L HEPES (pH 7.2), 137mM NaCl, 5mM CoCl2, 0.5mM MgCl2, 6mM DTT, 10mM dATP and 0.1mM CrCl2.

[0060] Experimental Example 2 Comparison of the TdT reaction buffer system for detecting cell apoptosis using the TUNEL technique of the present invention with that of mainstream competing products

[0061] Under the premise of ensuring that other components of the mainstream competitor are consistent, only the TdT reaction buffer system in the mainstream competitor is changed to the optimal TdT reaction buffer system for detecting cell apoptosis by TUNEL technology determined in Experimental Example 1. According to the "Steps for detecting cell apoptosis by TUNEL technology for alkyne-labeled dUTP", the apoptosis of HeLa cells was detected, and the cell nucleus was stained with Hoechst. The results are as follows Figure 3 As shown in the results, the addition of CrCl2 to the TdT reaction buffer system facilitates the binding of EdUTP / fluorescein-dUTP to the 3'-OH terminus, resulting in a strong fluorescence signal with no nonspecificity. Compared to mainstream competing products, this significantly reduces fluorescence background and nonspecificity, offering a clear advantage.

[0062] Experimental Example 3: The TdT reaction buffer system for detecting cell apoptosis using the TUNEL technique of the present invention is applied to the traditional TUNEL technique for detecting cell apoptosis

[0063] The TdT reaction buffer system of the traditional competitor was replaced with the optimal TdT reaction buffer system for TUNEL technique detection of cell apoptosis determined in Experimental Example 1. The apoptosis of HeLa cells was detected using the traditional TUNEL technique, and the cell nuclei were stained with Hoechst. The results are as follows: Figure 4 As shown, the TdT reaction buffer system for detecting cell apoptosis by the TUNEL technique of the present invention has a non-specific reduction and a signal enhancement compared with traditional competing products, indicating that it has advantages, indicating that the TdT reaction buffer system for detecting cell apoptosis by the TUNEL technique of the present invention is also suitable for detecting cell apoptosis by the traditional TUNEL technique.

[0064] Experimental Example 4 Effects of Different TdT Reaction Buffer Systems on TUNEL Detection of Cell Apoptosis

[0065] Under the premise of ensuring that other components of the mainstream competitor are the same, only the TdT reaction buffer system in the mainstream competitor is changed to the TdT reaction buffer system of combination 1-3. According to the "steps for detecting cell apoptosis by TUNEL technique for alkyne-labeled dUTP", the apoptosis of HeLa cells is detected. The results are as follows Figure 5 As shown, it is shown that the TdT reaction buffer system combination for detecting cell apoptosis using the TUNEL technique provided by the present invention has clean cell background, low nonspecificity and strong signal.

[0066] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. The application of CrCl2 in preparing TdT reaction buffer system is characterized in that: The concentration of CrCl2 is 0.01-0.5 mM.

2. The use according to claim 1, characterized in that The concentration of CrCl2 is 0.1 mM.

3. A TdT reaction buffer system, characterized in that: The TdT reaction buffer system consists of HEPES, NaCl, CoCl2, MgCl2, DTT, dATP and CrCl2; The concentration of CrCl2 is 0.01-0.5 mM; The concentration of HEPES is 10-60 mmol / L, and the pH is 7.2; the concentration of NaCl is 100-150 mM; the concentration of CoCl2 is 1-10 mM; the concentration of MgCl2 is 0.1-1 mM; the concentration of DTT is 2-10 mM; and the concentration of dATP is 1-20 mM.

4. The TdT reaction buffer system according to claim 3, characterized in that The concentration of CrCl2 is 0.1 mM.

5. A kit comprising the TdT reaction buffer system according to any one of claims 3 to 4.

6. Use of the TdT reaction buffer system according to any one of claims 3 to 4 or the kit according to claim 5 in detecting cell apoptosis using TUNEL technology, characterized in that: The application is for non-disease diagnosis or treatment purposes.

7. Use of the TdT reaction buffer system according to any one of claims 3 to 4 or the kit according to claim 5 in preparing a product for detecting cell apoptosis using the TUNEL technique.

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