Micronucleus purification kit and method
By providing a micronucleus purification kit and a simplified sucrose density gradient centrifugation technology, the complex and cumbersome micronucleus extraction process is solved, and efficient and simple micronucleus purification is achieved, which is suitable for clinical applications.
Patent Information
- Application Number
- CN202411935838.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-04-15
AI Technical Summary
The existing micronucleus extraction technology is complex and requires the preparation of multiple reagents. The operation is cumbersome and not suitable for clinical applications.
A micronucleus purification kit is provided, including cytochalasin B, buffer I, buffer II, lysate, supplementary solution I, supplementary solution II, sucrose I and sucrose II. The efficient purification of micronucleus is achieved through simplified solution preparation and sucrose density gradient centrifugation technology.
The micronucleus extraction process is simplified, the experimental time is reduced, and the efficiency and purity of purification and separation are improved, making it suitable for clinical applications.
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Figure CN119823981A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of biological detection, and in particular to a micronucleus purification kit and method. Background Art
[0002] Micronucleus (MCN), also called satellite nucleus, is an abnormal structure in eukaryotic cells and a manifestation of chromosomal aberration in interphase cells. Micronuclei are often produced by various physical and chemical factors, such as radiation and chemical agents, on dividing cells. Micronucleus testing is used in radiation damage, radiation protection, chemical mutagens, new drug trials, safety evaluation of food additives, as well as diagnosis of chromosomal genetic diseases and precancer. At present, micronucleus extraction involves many reagents, complex operations and requires high professional knowledge, which is not conducive to actual clinical applications. Summary of the invention
[0003] The embodiments of the present application provide a micronucleus purification kit and method, which effectively solve the pain point of complex solution preparation required in micronucleus extraction experiments, the overall process requires a short time, and the separation purity is high.
[0004] In a first aspect, an embodiment of the present application provides a micronucleus purification kit, characterized in that the kit comprises:
[0005] Cytochalasin B;
[0006] Buffer I; the buffer I comprises 1.8 M sucrose, 10 mM tris-HCl, 5 mM magnesium acetate and 0.1 mM ethylenediaminetetraacetic acid (EDTA), and the pH of the buffer I is 8.0;
[0007] Buffer II; the buffer II is specifically a frozen phosphate buffer PBS containing bovine serum albumin BSA with a volume concentration of 0.3%, ethylphenyl polyethylene glycol NP-40 with a volume concentration of 0.1%, and protease inhibitors;
[0008] Lysis solution; the lysate comprises 10 mM Tris-HCl, 2 mM magnesium acetate, 3 mM CaCl2, 0.32 M sucrose, 0.1 mM EDTA and a detergent, and the pH of the lysate is 8.5;
[0009] Supplement I: 1 mM dithiothreitol, 0.3% BSA, 0.15 mM spermine, 0.75 mM sucrose and protease inhibitors;
[0010] Supplement II: 1 mM dithiothreitol, 0.3% BSA, 0.15 mM spermine, and 0.75 mM spermidine;
[0011] Sucrose I;
[0012] Sucrose II;
[0013] The weights of sucrose I and sucrose II are different to form a sucrose density gradient.
[0014] In some embodiments, for 50 servings of the micronucleus purification kit, the weight of the cytochalasin B is 10 mg, the volume of the buffer I is 50 mL, the volume of the buffer II is 40 mL, the volume of the lysate is 50 mL, the volume of the supplement I is 5 mL, the volume of the supplement II is 5 mL, the weight of the sucrose I is 12.323 g; the weight of sucrose II is 8.215 g.
[0015] In some embodiments, the detergent is 0.1% by volume NP-40; or, the detergent is Triton X-100.
[0016] In some embodiments, the cytochalasin B is stored at -20°C, and the rest is stored at 4°C.
[0017] In a second aspect, an embodiment of the present application provides a micronucleus purification method, which is applied to the micronucleus purification kit as described in the first aspect, and the method comprises:
[0018] Step S1: Dissolve cytochalasin B with 500 μL of pure water and store at -20°C; dilute 1 mL of lysis solution to 10 mL to form lysis dilution solution; dilute 1 mL of buffer I to 10 mL to form buffer dilution solution I; dilute 800 μL of buffer II to 40 mL to form buffer dilution solution II; dilute two portions of sucrose I with pure water to 40 mL to form sucrose dilution solution I; dilute two portions of sucrose II with pure water to 30 mL to form sucrose dilution solution II;
[0019] Step S2: adding 10 μL of cytochalasin B to 20 mL of the sample cell solution, and incubating to form an initial incubation solution;
[0020] Step S3: centrifuging the initial incubation solution, adding 10 mL of the lysis diluent to the precipitate produced by centrifugation, and lysing on ice for at least 30 minutes;
[0021] Step S4: adding 100 μL of supplement solution I to the generated post-lysis solution, and mixing to generate a lysate solution;
[0022] Step S5: adding 100 μL of supplement solution II to 10 mL of buffer diluent I, and mixing the lysate solution therewith to obtain a lysate mixture;
[0023] Step S6: using sucrose diluent I and sucrose diluent II to form a sucrose density gradient, and using the sucrose density gradient to extract the micronuclei in the lysis mixture by centrifugation and layered collection.
[0024] In some embodiments, step S6 specifically includes:
[0025] S61: adding 100 μL of supplement solution II to 10 ml of buffer diluent I, and mixing the lysis mixture therewith to obtain a lysis mixture;
[0026] S62: Take a conical tube, add 40 mL of sucrose diluent I, then add 20 mL of lysis mixture, and finally add 30 mL of sucrose diluent II, and centrifuge at 950g and 4°C for 20 minutes;
[0027] S63: After centrifugation, discard the top fraction, collect the next target fraction, add 40 mL of buffer diluent II, centrifuge and discard the supernatant to obtain the micronucleus precipitate.
[0028] In some embodiments, the volume of the top fraction is 4 mL; the volume of the target fraction is 10 mL.
[0029] In some embodiments, for step S2, the incubation operation is specifically incubating at 37°C for 45 minutes, and for step S4, the mixing operation is homogenizing 10 times.
[0030] In some embodiments, the method further comprises:
[0031] S0: Form a sample cell solution; expand the cell sample in a culture dish, then collect the cells after trypsin digestion; resuspend the collected cells in culture medium at a concentration of about 5×10 6 Pieces / mL;
[0032] The cell sample is produced by the parental cell line HEK293T or HeLa, and the culture medium is a DMEM high-glucose culture medium supplemented with 10% Australian fetal bovine serum FBS and 1% penicillin or streptomycin.
[0033] In some embodiments, steps S4 to S6 should all be performed on ice.
[0034] The embodiments of the present application provide a micronucleus purification kit and method. The reagents in the kit have been recombined and concentrated in multiples. The user does not need to purchase and prepare the required solutions separately, which effectively solves the pain point of complex solution preparation required in micronucleus extraction experiments, is convenient and time-saving, and has a high extraction purity. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Figure 1A schematic flow chart of a micronucleus extraction method provided in the present application is shown. DETAILED DESCRIPTION
[0036] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present application. It is to be understood that the specific embodiments described herein are only used to explain the related applications, rather than to limit the present application. It should also be noted that, for ease of description, only the parts related to the related applications are shown in the accompanying drawings.
[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application.
[0038] In the following description, reference is made to “some embodiments”, which describe a subset of all possible embodiments, but it will be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.
[0039] The present application provides a micronucleus purification reagent and method. The basic principle is to lyse the cells of the isolated sample containing micronuclei with a lysis solution, separate the micronuclei using a sucrose density gradient, and purify the micronuclei by centrifugation, layered collection and other operations.
[0040] The embodiments of the present application will be described in detail below with reference to the accompanying drawings.
[0041] In one embodiment, a micronucleus purification reagent is provided, the micronucleus purification reagent comprising:
[0042] (a) Cytochalasin B;
[0043] (b) buffer I; buffer I comprises 1.8 M sucrose, 10 mM Tris-HCl, 5 mM magnesium acetate and 0.1 mM EDTA, and the pH of buffer I is 8.0;
[0044] (c) buffer II; buffer II specifically comprises a chilled phosphate buffer saline (PBS) containing 0.3% by volume of bovine serum albumin (BSA), 0.1% by volume of ethylphenyl polyethylene glycol NP-40, and a protease inhibitor;
[0045] (d) lysis solution; the lysis solution comprises 10 mM Tris-HCl, 2 mM magnesium acetate, 3 mM CaCl2, 0.32 M sucrose, 0.1 mM EDTA and a detergent, and the pH of the lysis solution is 8.5;
[0046] (e) Supplement I: 1 mM dithiothreitol, 0.3% BSA, 0.15 mM spermine, 0.75 mM sucrose and protease inhibitors;
[0047] (f) Supplement II: 1 mM dithiothreitol, 0.3% BSA, 0.15 mM spermine, and 0.75 mM spermidine;
[0048] (g) sucrose I;
[0049] (i) Sucrose II;
[0050] The weights of sucrose I and sucrose II are different to form a sucrose density gradient.
[0051] It should be noted that protease inhibitors are generally mixtures. There are now commercial protease inhibitors, which can be diluted according to the dilution multiple indicated on the product. For example, if the product is 100×, it should be diluted 1:100 when used.
[0052] Thus, the embodiment of the present application provides a systematic reagent, provides a concentrated solution of the reagents required in the whole process of micronucleus purification, selects the best combination and the most suitable ratio, is simple and convenient, has low professional requirements for technicians, and is easy to promote and apply.
[0053] In some embodiments, each micronucleus purification reagent can be used for testing of 50 people, wherein, for each micronucleus purification reagent, the weight of cytochalasin B is 10 mg, the volume of buffer I is 50 mL, the volume of buffer II is 40 mL, the volume of lysate is 50 mL, the volume of supplementary solution I is 5 mL, the volume of supplementary solution II is 5 mL, the weight of sucrose I is 12.323 g; the weight of sucrose II is 8.215 g.
[0054] In this way, the reagents in the micronucleus purification kit provided in the embodiment of the present application have been concentrated multiple times, and the user does not need to purchase and configure the required solutions additionally, which well solves the pain point of complex solution configuration required in the micronucleus extraction experiment, saving time and effort.
[0055] In some embodiments, the detergent is 0.1% by volume NP-40; or, the detergent is Triton X-100.
[0056] In some embodiments, cytochalasin B is stored at -20°C and the rest is stored at 4°C.
[0057] In summary, the present application provides a systematic kit required for micronucleus purification, specifically providing concentrated solutions of reagents required for each step. The experimental steps are clear and definite, do not rely on ultracentrifuges, and are simple and convenient.
[0058] In another embodiment of the present application, see Figure 1 , which shows a schematic flow diagram of a micronucleus purification method provided in an embodiment of the present application, wherein the micronucleus purification method uses the aforementioned micronucleus purification kit to extract micronuclei. Figure 1 As shown, the method specifically includes:
[0059] Step S1: Dissolve cytochalasin B with 500 μL of pure water and store at -20°C; dilute 1 mL of lysis solution to 10 mL to form lysis dilution solution; dilute 1 mL of buffer I to 10 mL to form buffer dilution solution I; dilute 800 μL of buffer II to 40 mL to form buffer dilution solution II; dilute two portions of sucrose I with pure water to 40 mL to form sucrose dilution solution I; dilute two portions of sucrose II with pure water to 30 mL to form sucrose dilution solution II;
[0060] Step S2: adding 10 μL of cytochalasin B to 20 mL of the sample cell solution, and incubating to form an initial incubation solution;
[0061] Step S3: centrifuge the initial incubation solution, add 10 mL of lysis diluent to the precipitate produced by centrifugation, and lyse on ice for at least 30 minutes;
[0062] Step S4: adding 100 μL of supplement solution I to the generated post-lysis solution, and mixing to generate a lysate solution;
[0063] Step S5: adding 100 μL of supplement solution II to 10 mL of buffer diluent I, and mixing the lysate solution therewith to obtain a lysate mixture;
[0064] Step S6: using sucrose diluent I and sucrose diluent II to form a sucrose density gradient, and using the sucrose density gradient to extract the micronuclei in the lysis mixture by centrifugation and layered collection.
[0065] In this way, the solutions required for each step in the micronucleus purification process can be prepared through the purification kit, and the technicians can configure and purify according to the instructions. It is convenient and time-saving. The purification can be completed in only 1.5 hours. It does not rely on an ultracentrifuge and is easy to implement and promote.
[0066] In some embodiments, step S6 specifically includes:
[0067] S61: adding 100 μL of supplement solution II to 10 ml of buffer diluent I, and mixing the lysis mixture therewith to obtain a lysis mixture;
[0068] S62: Take a conical tube, add 40 mL of sucrose diluent I, then add 20 mL of lysis mixture, and finally add 30 mL of sucrose diluent II, and centrifuge at 950g and 4°C for 20 minutes;
[0069] S63: After centrifugation, discard the top fraction, collect the next target fraction, add 40 mL of buffer diluent II, centrifuge and discard the supernatant to obtain the micronucleus precipitate.
[0070] In some embodiments, the volume of the top fraction is 4 mL; the volume of the target fraction is 10 mL.
[0071] Of course, the volumes of the top fraction and the target fraction can be adjusted according to the actual application scenario.
[0072] In some embodiments, for step S2, the incubation operation is specifically incubating at 37°C for 45 minutes, and for step S4, the mixing operation is homogenizing 10 times.
[0073] In some embodiments, the method further comprises:
[0074] S0: Form a sample cell solution; expand the cell sample in a culture dish, then collect the cells after trypsin digestion; resuspend the collected cells in culture medium at a concentration of about 5×10 6 Pieces / mL;
[0075] The cell samples were produced by the parental cell lines HEK293T or HeLa, and the culture medium was DMEM high-glucose medium supplemented with 10% Australian fetal bovine serum FBS and 1% penicillin or streptomycin.
[0076] It should be noted that if the cell sample is produced by other cell lines, the specific parameters and dosages in the above steps can be adjusted through experiments to achieve the best purification effect.
[0077] In some embodiments, steps S4 to S6 should all be performed on ice.
[0078] Thus, the micronucleus purification method provided in the embodiment of the present application has a simple process, a short time required, a good purification effect, and is easy to promote clinically.
[0079] In yet another embodiment, a micronucleus purification kit for 50 people and a method for using the same are provided.
[0080] First, this kit consists of the components shown in Table 1.
[0081] Table 1
[0082]
[0083]
[0084] Secondly, the specific components of each reagent in the micronucleus purification kit are shown in Table 2.
[0085] Table 2
[0086]
[0087] The specific steps of purifying micronuclei using the above-mentioned micronuclei purification kit are as follows:
[0088] (1) Dissolve cytochalasin B in 500 μL of pure water before use and store at -20°C.
[0089] (2) Collecting and resuspending sample cells: The sample cells were expanded in a 245×245×25 mm culture dish, and then digested with trypsin and collected. The collected sample cells were resuspended in culture medium at a concentration of approximately 5×10 6 / mL, forming a sample cell solution;
[0090] The amount of drugs required in the following operations should be calculated based on the number of cells collected. For example, to resuspend cells in 20 mL of culture medium, the following amount of drugs is required:
[0091] Pipette 1 mL of 10× lysis buffer and dilute to 10 mL to form lysis dilution buffer;
[0092] Pipette 1mL 10×BufferⅠ and dilute to 10mL to form buffer diluent I
[0093] Pipette 800 μL of 50× Buffer II and dilute to 40 mL to form Buffer Diluent II;
[0094] Take two portions of sucrose 1 and dilute them in 40 mL of pure water to form sucrose dilution I;
[0095] Take two portions of sucrose 2 and dilute them in 30 mL of pure water to form sucrose dilution II.
[0096] After that, the specific steps are as follows:
[0097] (3) Pretreatment: Add 10 μL of cytochalasin B and incubate at 37°C for 45 min.
[0098] (4) Re-incubation: Centrifuge the cells at 300 g for 5 minutes, discard the supernatant, add 10 mL of lysis buffer, gently pipette to resuspend the cells, and lyse on ice for at least 30 minutes.
[0099] (5) Add 100 μL of Supplementary Diluent I to the Lysis Diluent, mix well, and homogenize the Lysis Diluent up and down 10 times.
[0100] (6) Add 100 μL of Supplementary Diluent II to 10 ml of Buffer Diluent I and mix the lysate with it to obtain a lysis mixture. Take a 100 ml conical tube, add 40 ml of Sucrose Diluent I, then add 20 ml of the lysis mixture, and finally add 30 ml of Sucrose Diluent II, and centrifuge at 950g and 4°C for 20 minutes.
[0101] (7) After centrifugation, discard the 4 ml top fraction, collect the next 10 ml, add 40 ml of buffer diluent II, and centrifuge at 8000 rpm and 4°C for 20 minutes. Discard the supernatant and collect the micronucleus precipitate for the next experiment.
[0102] It should be noted that all work should be performed on ice, starting from resuspending cells in lysis diluent.
[0103] In addition, the 1.8 M sucrose dilution should be pipetted gently to allow it to mix properly with the 1.6 M sucrose dilution to form a density gradient.
[0104] The volumes described for the sucrose density gradient are optimal for a 10 mL mixture of lysate and sucrose; if the mixture volume is different, the amount of sucrose dilution should be adjusted accordingly.
[0105] Do not invert the conical tube containing the sucrose density gradient to avoid disturbing the density gradient.
[0106] For the above embodiment, the volume of the collected layers is optimal for the volume of the above mixture and the sucrose diluent. If a sucrose concentration gradient of different ratios is used and the collected portion after centrifugation does not contain the expected particles, the collected volume should be adjusted accordingly.
[0107] When verifying the efficiency of cell lysis under a microscope, if the primary nuclei and micronuclei are still wrapped by the cell membrane, if a different cell line than HEK293T or HeLa is used, the choice of detergent or the final concentration in the lysis buffer may be inappropriate. You can use another detergent (such as Triton X-100) or try increasing the concentration of NP-40.
[0108] If micronuclei are not separated from the primary nuclei, increase the number of homogenizations. Omitting the cytochalasin B treatment may also result in the inability to separate micronuclei from the primary nuclei.
[0109] Details not disclosed in the embodiments of the present application may be understood by referring to the description of the aforementioned embodiments.
[0110] The above are only preferred embodiments of the present application and are not intended to limit the protection scope of the present application.
[0111] It should be noted that, in this application, the terms "include", "comprises" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "includes a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.
[0112] The serial numbers of the above-mentioned embodiments of the present application are for description only and do not represent the advantages or disadvantages of the embodiments.
[0113] The methods disclosed in several method embodiments provided in this application can be arbitrarily combined without conflict to obtain new method embodiments.
[0114] The features disclosed in several product embodiments provided in this application can be arbitrarily combined without conflict to obtain new product embodiments.
[0115] The features disclosed in several method or device embodiments provided in this application can be arbitrarily combined without conflict to obtain new method embodiments or device embodiments.
[0116] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.
Claims
1. A micronucleus purification kit, characterized in that: The kit comprises: Cytochalasin B; Buffer I; the buffer I comprises 1.8 M sucrose, 10 mM tris-HCl, 5 mM magnesium acetate and 0.1 mM ethylenediaminetetraacetic acid (EDTA), and the pH of the buffer I is 8.0; Buffer II; the buffer II is specifically a frozen phosphate buffer PBS containing bovine serum albumin BSA with a volume concentration of 0.3%, ethylphenyl polyethylene glycol NP-40 with a volume concentration of 0.1%, and protease inhibitors; Lysis solution; the lysate comprises 10 mM Tris-HCl, 2 mM magnesium acetate, 3 mM CaCl2, 0.32 M sucrose, 0.1 mM EDTA and a detergent, and the pH of the lysate is 8.5; Supplement I: 1 mM dithiothreitol, 0.3% BSA, 0.15 mM spermine, 0.75 mM sucrose and protease inhibitors; Supplement II: 1 mM dithiothreitol, 0.3% BSA, 0.15 mM spermine, and 0.75 mM spermidine; Sucrose I; Sucrose II; The weights of sucrose I and sucrose II are different to form a sucrose density gradient.
2. The micronucleus purification kit according to claim 1, characterized in that For the micronucleus purification kit for 50 people, the weight of the cytochalasin B is 10 mg, the volume of the buffer I is 50 mL, the volume of the buffer II is 40 mL, the volume of the lysate is 50 mL, the volume of the supplement I is 5 mL, the volume of the supplement II is 5 mL, the weight of the sucrose I is 12.323 g; the weight of sucrose II is 8.215 g.
3. The micronucleus purification kit according to claim 1, characterized in that The detergent is NP-40 with a volume ratio of 0.1%; or, the detergent is Triton X-100.
4. The micronucleus purification kit according to any one of claims 1 to 3, characterized in that The cytochalasin B was stored at -20°C, and the rest was stored at 4°C.
5. A method for purifying micronuclei, characterized in that: Applicable to the micronucleus purification kit according to any one of claims 1 to 4, the method comprising: Step S1: Dissolve cytochalasin B with 500 μL of pure water and store at -20°C; dilute 1 mL of lysis solution to 10 mL to form lysis dilution solution; dilute 1 mL of buffer I to 10 mL to form buffer dilution solution I; dilute 800 μL of buffer II to 40 mL to form buffer dilution solution II; dilute two portions of sucrose I with pure water to 40 mL to form sucrose dilution solution I; dilute two portions of sucrose II with pure water to 30 mL to form sucrose dilution solution II; Step S2: adding 10 μL of cytochalasin B to 20 mL of the sample cell solution, and incubating to form an initial incubation solution; Step S3: centrifuging the initial incubation solution, adding 10 mL of the lysis diluent to the precipitate produced by centrifugation, and lysing on ice for at least 30 minutes; Step S4: adding 100 μL of supplement solution I to the generated post-lysis solution, and mixing to generate a lysate solution; Step S5: adding 100 μL of supplement solution II to 10 mL of buffer diluent I, and mixing the lysate solution therewith to obtain a lysate mixture; Step S6: using sucrose diluent I and sucrose diluent II to form a sucrose density gradient, and using the sucrose density gradient to extract the micronuclei in the lysis mixture by centrifugation and layered collection.
6. The method according to claim 5, characterized in that The step S6 specifically includes: S61: adding 100 μL of supplement solution II to 10 ml of the buffer diluent I, and mixing the lysis mixture therewith to obtain a lysis mixture; S62: Take a conical tube, add 40 mL of the sucrose diluent I, then add 20 mL of the lysis mixture, and finally add 30 mL of sucrose diluent II, and centrifuge at 950 g and 4° C. for 20 minutes; S63: After centrifugation, the top fraction is discarded, the next target fraction is collected, 40 mL of the buffer diluent II is added, and the micronucleus precipitate is obtained after centrifugation and the supernatant is discarded.
7. The method according to claim 6, characterized in that The volume of the top fraction was 4 mL; the volume of the target fraction was 10 mL.
8. The method according to claim 6, characterized in that For step S2, the incubation operation is specifically incubating at 37° C. for 45 minutes. For step S4, the mixing operation is 10 times of homogenization.
9. The method according to claim 5, characterized in that The method further comprises: S0: The cell sample was expanded in a culture dish, and then the cells were collected after trypsin digestion; the collected cells were resuspended in culture medium at a concentration of about 5×10 6 Pieces / mL; The cell sample is produced by the parental cell line HEK293T or HeLa, and the culture medium is a DMEM high-glucose culture medium supplemented with 10% Australian fetal bovine serum FBS and 1% penicillin or streptomycin.
10. The method according to claim 5, characterized in that The steps S4 to S6 should all be performed on ice.