CN117054200A - TUNEL special-purpose fixed rupture membrane agent for flow cytometry analysis of apoptotic cells, preparation method and application thereof - Google Patents

Abstract

The application provides a TUNEL special-purpose fixed rupture membrane agent for analyzing apoptotic cells by flow cytometry, and a preparation method and application thereof. The fixed membrane rupture agent comprises a fixed liquid and a membrane rupture liquid; the fixing solution comprises glutaraldehyde, paraformaldehyde, methanol, glycerol, triton X-100, sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate and the balance of water; the membrane rupture liquid comprises NP40, triton X-100, BSA, sodium chloride, potassium dihydrogen phosphate, trisodium citrate dihydrate and the balance of water. The TUNEL detection method is suitable for TUNEL detection of suspension cells, single cell suspension, adherent cells and primary cells, positive signals are clear, the resolution of positive groups and negative groups is high, and the experimental universality is strong; the method can also permeate the nuclear membrane, promote TUNEL marking liquid to smoothly enter the cell nucleus, effectively mark broken DNA, and improve the repetition rate and success rate of TUNEL experiments.

Description

TUNEL's special fixed membrane-breaking agent for analyzing apoptotic cells by flow cytometry and its Preparation methods and applications

Technical field

The invention belongs to the technical field of flow cytometry, and specifically relates to TUNEL's special fixed membrane-breaking agent for analyzing apoptotic cells by flow cytometry and its preparation method and application.

Background technique

When cells undergo apoptosis, they activate some specific DNA endonucleases, which cut the genomic DNA between nucleosomes. The DNA of apoptotic cells is cut into 180-200bp fragments. After the cells are fixed and permeabilized, TdT enzyme (terminal deoxyribonucleic acid transferase) connects the fluorescently labeled dUTP to the exposed 3'-OH end of the broken DNA of the cells. Add fluorescent dUTP to these ends to label late-stage apoptotic cells, so that apoptotic cells become fluorescent, and then apoptotic cells with DNA fragmentation can be detected through a fluorescence detection instrument.

TUNEL is the gold standard for apoptosis detection. It is divided into two types according to different detection instruments. One is in situ TUNEL detection, which mainly takes pictures and collects the fluorescent signals of apoptotic cells through a fluorescence microscope. It can be applied to paraffin sections. , frozen sections, cell slides and other samples; one is flow cytometry TUNEL detection, which mainly uses flow cytometry to detect single cell suspensions (such as digested adherent cells, suspended cells, tissue, etc., single cell suspensions) The fluorescence signals of apoptotic cells were collected and analyzed.

Both detection methods have their own advantages and disadvantages. TUNEL, which is used for in-situ detection, can intuitively observe the subcellular location of apoptosis genes from the subcellular structure of cells, but it cannot solve the quantitative analysis of apoptotic cells, while cell slides The problem of apoptotic cells easily falling off in in-situ detection cannot be solved, especially for some cells that are difficult to adhere to. After the in-situ detection process, too many cells fall off, resulting in inaccurate detection results and experimental failure, and suspension cells cannot be solved. TUNEL detection; streaming TUNEL can solve this problem.

The flow TUNEL detection kit currently on the market only contains dyeing labeling components such as dyeing labeling solution, equilibrium solution, and TDT enzyme. However, the details of the processing of cell samples are vague, and the processing of cell samples is the key to the success or failure of TUNEL flow cytometry. We collected the fixatives and membrane-breaking solutions used in flow cytometry on the market. After testing, these fixatives and membrane-breaking solutions It can be used for the fixation and membrane rupture of cell protein antigens detected by other flow cytometry methods, but it cannot meet the fixation and membrane rupture requirements of the TUNEL method for detection of fragmented DNA.

Patent CN115754305A reports a cell-fixing membrane-breaking agent specifically used for flow cytometry detection and its use method. The disclosed fixed membrane-breaking solution is mainly used for flow cytometry to detect intracellular protein antigens and antibodies. After testing, the fixed membrane-breaking solution is not suitable for the environment where TUNEL flow cytometry detects broken DNA to label apoptotic cells.

Patent CN112229696A also reports a cell fixative for flow cytometry and its use method. Its application is also mainly used for flow cytometry to detect protein antigens and antibodies in cells. After testing, the fixed membrane-breaking solution also It is not suitable for environments where TUNEL flow cytometry detects fragmented DNA to label apoptotic cells.

As the first step in the experiment, sample processing plays a decisive role. TUNEL detection requires TDT enzyme and labeling solution to penetrate the outer cell membrane and nuclear membrane, and complete labeling at the level of DNA in the nucleus. Currently, there are many methods on the market. Fixatives and rupture fluids, as well as related fixative rupture fluid patents, are all antigen-antibody reactions for protein detection in the cytoplasm and are not suitable for DNA labeling in the nucleus. To meet the specific needs of TUNEL experiments, a solution needs to be developed. A set of special fixed membrane-breaking fluid that can be used for TUNEL detection of DNA markers to improve the success rate and sensitivity of TUNEL experiments.

Contents of the invention

The purpose of the present invention is to provide a TUNEL-specific fixed membrane-breaking agent for flow cytometry analysis of apoptotic cells and its preparation method and application in view of the above-mentioned shortcomings of the existing technology.

In order to achieve the above objects, the present invention adopts the following technical solutions:

The first object of the present invention is to provide a TUNEL-specific fixed membrane-breaking agent for flow cytometric analysis of apoptotic cells. The fixed membrane-breaking liquid includes a fixative and a membrane-breaking liquid; in terms of weight percentage, the fixed membrane-breaking agent The membrane-breaking solution includes a fixative and a membrane-breaking solution; in terms of weight percentage, the fixative includes glutaraldehyde 0.1 to 5%, paraformaldehyde 1% to 25%, methanol 0.5% to 10%, and glycerin 0.5% to 10%. %, Triton 2% and the balance is water; in terms of weight percentage, the membrane-breaking solution includes NP40 0.02% ~ 3%, Triton X-100 0.01% ~ 3%, BSA 0.2% ~ 10%, sodium chloride 0.1% ~ 3%, potassium chloride 0.1% to 1%, potassium dihydrogen phosphate 0.1% to 1%, trisodium citrate dihydrate 0.1% to 2% and the balance is water.

Further, the pH of the fixative solution is 7-7.6, and the pH of the membrane-breaking solution is 7-7.6.

The second object of the present invention is to provide a method for preparing the above-mentioned fixative, which includes the following steps: dissolving paraformaldehyde in pure water, and then weighing and adding sodium chloride, potassium chloride, and diphosphate in sequence. Potassium hydrogen and disodium hydrogen phosphate dodecahydrate were stirred and dissolved at room temperature. Adjust the pH to 7.0~7.6, then add the methanol solution and stir while adding. After the solution is mixed, add glutaraldehyde liquid, glycerin and Triton X-100. , stir and mix at room temperature, let it stand to eliminate bubbles and then adjust to volume to obtain the fixative.

The third object of the present invention is to provide the preparation method of the above-mentioned membrane-breaking solution, which includes the following steps: sequentially weigh sodium chloride, potassium chloride, potassium dihydrogen phosphate and trisodium citrate dihydrate, add pure water, and cool at room temperature. Stir and mix, adjust the pH to 7.0~7.6, weigh and add NP40, stir and mix, add Triton Dilute to 1L, aliquot and store at -20°C to obtain the membrane-breaking solution.

The fourth purpose of this application is to provide the application of the above-mentioned TUNEL-specific fixed membrane-breaking agent in detecting apoptosis of cell samples by TUNEL method.

Further, the cell sample includes any one of suspension cells, single cell suspension, adherent cells and primary cells.

Furthermore, it is used to break the nuclear membrane of the cell nucleus and stain the DNA in the nucleus.

The fifth purpose of this application is to provide the above-mentioned TUNEL-specific fixed membrane-breaking solution method for detecting apoptosis-broken DNA, including the following specific steps:

S1. Collect the cell sample, add PBS buffer to resuspend the cell pellet, and then add the fixative for fixation;

S2. Centrifuge the fixed cells, discard the supernatant, add the membrane rupture solution, resuspend the cell pellet for membrane rupture, terminate the membrane rupture, centrifuge again, and discard the supernatant;

S3. Stain the cells after membrane rupture according to the TUNEL method and use the machine for detection.

Further, the number of collected cell samples is 1 to 5 × 10 ⁶ per detection tube. Use 100 to 600 μL of PBS to resuspend the cell pellet and mix well. Add a fixative in a volume of 100 to 600 μL, and the fixation time is 45 to 60 minutes, fixed condition is room temperature.

Further, add 100 to 600 μL of membrane rupture solution to the centrifuged cells, resuspend the cell pellet and mix well. The membrane rupture conditions are 5 to 20 minutes on ice. After membrane rupture, 1 to 2 mL of PBS buffer needs to be added for rupture. Membranes were terminated in PBS buffer containing 1% BSA.

Compared with the existing technology, the beneficial effects brought by the technical solution provided by the present invention are:

(1) The present invention uses phosphate buffer as an isotonic solution to effectively reduce the surface tension of cells and maintain the morphology of cells. Paraformaldehyde-fixed cells can form aldehyde cross-links between cell proteins, but for nucleic acids and sugars The fixation effect is poor, so that the shape and boundaries of the nucleus and organelles are unclear and shrinkage occurs. Glutaraldehyde reagent is added to this fixative, and the method of co-fixation with paraformaldehyde can be used to fix DNA and sugars at the same time. Make the boundaries of the cell nucleus clearer and more conducive to DNA labeling and staining; in order to give full play to the role of the membrane-breaking solution in breaking the nuclear membrane in the next step, add Triton X-100 to the fixative while fixing the cells so that the cells are fixed while Make holes in the cell membrane to ensure that the outer cell membrane is fully permeable; add a small amount of methanol solution to maintain the stability of formaldehyde and glutaraldehyde and avoid deformation and polymerization of the fixative; add glycerol to ensure the stability of the fixative during long-term low-temperature storage; - Low-temperature storage at 20°C reduces the evaporation of reagent components and ensures the storage stability of the fixative.

(2) The present invention uses citrate buffer as an isotonic solution, which has an antibacterial effect on fixed cells and reduces the influence of metal ions; NP-40 and Triton X-100 are added to break the membrane together, and the membrane is broken in the fixative solution On the basis of the outer membrane of the cell, it further enters the cytoplasm, improving the efficiency of punching holes in the nuclear membrane, ensuring that the nuclear membrane in the cell is fully permeable, and ensuring that the TUNEL reaction solution fully enters the nuclear membrane for effective labeling of nuclear DNA; adding As a protein carrier for cells, BSA increases the surface tension of the liquid, reduces the impact of centrifugal pressure during centrifugation, and reduces cell loss; low-temperature storage at -20°C reduces the evaporation of reagent components and ensures the stability of the membrane-breaking solution.

(3) The fixative and membrane-breaking liquid provided by the present invention are suitable for TUNEL detection of suspended cells and single-cell suspensions, optimizing the conditions and time of fixation, optimizing the conditions and time of membrane rupture, and reducing DNA fragmentation in TUNEL detection. The positive signal is clear, the resolution of the positive group and the negative group is high, and the experiment is versatile.

(4) The fixative and membrane-breaking solution provided by the present invention are suitable for TUNEL detection of adherent cells. The positive signals are clear and the resolution of positive and negative groups is high, which can solve the TUNEL apoptosis detection of cells that are not firmly adherent.

(5) The fixative and membrane-breaking fluid provided by the present invention are suitable for TUNEL detection of primary cells, and can be co-stained with other phenotypic antibodies without affecting the phenotypic grouping results.

(6) The fixed membrane-breaking solution solution provided by the present invention can effectively permeate the outer membrane of the cell and further permeate the nuclear membrane, prompting the TUNEL labeling solution to smoothly enter the cell nucleus, effectively labeling the broken DNA, and improving the efficiency of the TUNEL experiment. Repeatability and success rates.

Description of the drawings

Figure 1 is a comparison chart of the apoptosis ratio results of detecting the apoptosis ratio of HL-60 cells between the fixed membrane-breaking fluid provided in Example 1 and Example 2 and unfixed living cells: compared with unfixed living cells, the fixed membrane-breaking solution provided in Example 1 Both the membrane rupture solution and the fixed membrane rupture solution in Example 2 resulted in significantly reduced cell morphology, lower FSC value, greater cell loss, lower yield, and no apoptotic signal was detected. This fixed membrane rupture solution is not suitable for TUNEL. Detection of labeled DNA;

Figure 2 is a comparison chart of the apoptosis results of mouse spleen cells verified by the fixed membrane-breaking fluid provided in Example 2, Example 4 and Example 5: the fixation effect of Example 4 is better than that of Example 5, but the apoptotic cells detected The negative group and positive group classification effect is poor; the fixed membrane rupture scheme of Example 4 leads to false positives in the sample and is not suitable for the detection of TUNEL-labeled DNA;

Figure 3 is a comparison chart of the apoptosis ratio of Jurkat cells detected in fixed membrane-breaking solutions provided in Example 7, Example 8 and Example 9: neither the proportion of formaldehyde nor paraformaldehyde in the fixed solution can improve the cell yield in the final product. , adding glutaraldehyde is beneficial to increase cell yield and cell status in the FSC chart. There are more cell debris, and membrane rupture conditions need to be optimized;

Figure 4 is a comparison chart of the results of optimizing the membrane rupture conditions: extending the fixation time to 60 minutes is beneficial to improving cell yield and maintaining cell morphology; optimizing the membrane rupture time to 10 minutes on ice is beneficial to reducing cell debris, but it still cannot be effectively destroyed. nuclear membrane;

Figure 5 shows the results of optimizing the composition of the fixative solution to detect the apoptosis ratio of Jurkat cells: increasing the membrane-breaking component in the fixative and extending the fixation time to 60 minutes will help increase the cell yield and maintain cell morphology; optimizing the composition of the membrane-breaking solution , after adding the protective effect of BSA, adjust the membrane rupture time to 5 minutes on ice, which will help reduce cell debris and can effectively rupture the nuclear membrane, but the grouping effect is slightly poor;

Figure 6 is a comparison chart of optimizing the rupture time of the membrane rupture solution and examining the results of different membrane rupture times: maintaining the fixation time of the fixative solution to 60 minutes is beneficial to increasing the cell yield and maintaining cell morphology; optimizing the composition of the membrane rupture solution and adding BSA After the protective effect, adjusting the membrane rupture time to 10, 20, and 30 minutes on ice can effectively break the nuclear membrane. From the analysis of the proportion of positive groups and average fluorescence brightness, the membrane rupture time of 10 minutes on ice has the best effect;

Figure 7 shows the results of verifying the effective concentration range of each reagent component of the fixed membrane-breaking solution and detecting the apoptosis ratio of HL-60 cells and Jurkat cells: adjust the ratio of each reagent within a certain range, and maintain the fixation time of the fixative solution to room temperature for 60 minutes. , adjust the membrane rupture time to 10 minutes on ice, which can effectively rupture the nuclear membrane and detect the apoptosis ratio of Jurkat cells and HL-60 cells;

Figure 8 is a graph showing the results of verifying the effective concentration range of each reagent component of the fixation rupture solution and detecting the apoptosis ratio of adherent cells Hela cells: adjust the ratio of each reagent within a certain range: maintain the fixation time of the fixation solution to room temperature for 60 minutes, adjust The membrane rupture time is 10 minutes on ice, which can effectively detect the apoptosis ratio of adherent HeLa cells;

Figure 9 shows the results of the special TUNEL fixation rupture solution to detect the apoptosis ratio of mouse spleen primary cells: maintain the fixation time of the fixative to room temperature for 60 minutes, adjust the rupture time to 10 minutes on ice, and the primary cells can be effectively detected Cell apoptosis ratio in mouse spleen single cell suspension, and can be co-stained with other phenotypic staining antibodies without affecting the staining results;

Figure 10 is a special TUNEL fixed membrane rupture solution to detect the apoptosis ratio of Jurkat labeled with different fluorescently labeled dyes: maintain the fixation time of the fixative to room temperature for 60 minutes, adjust the membrane rupture time to 10 minutes on ice, and you can detect different fluorescent channels. Labeling solution, the results of the detected cell apoptosis ratio were consistent.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the specific implementation modes of the present invention will be further described in detail below with reference to specific embodiments and drawings.

Unless otherwise specified, the reagents and biological raw materials used in the examples of the present invention were purchased through commercial channels. The Chinese and English/abbreviations of the present invention are as follows:

Triton X-100: polyethylene glycol octylphenyl ether;

NP40: Polysorbate 40;

PBS buffer: phosphate buffered saline solution;

BSA: bovine serum albumin;

FSC value: forward angle scatter value measured by flow cytometry, representing cell size.

TUNEL assay: terminal deoxyribonucleotide transferase-mediated nick end labeling method.

Example 1:

This embodiment includes a cell fixative with the following formula:

Sodium dihydrogen phosphate 0.12%;

Disodium hydrogen phosphate 0.14%;

Sodium chloride 0.88%;

Paraformaldehyde 4%;

This embodiment includes a cell membrane-breaking solution with the following formula:

Sodium dihydrogen phosphate 0.12%;

Disodium hydrogen phosphate 0.14%;

Sodium chloride 0.88%;

Saponin 0.3%;

NP40 0.2%;

Tween20 0.2%;

BSA 1%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at room temperature.

Example 2

This embodiment includes a cell fixative with the following formula:

Absolute ethanol 70%;

Pure water 30%;

Measure 70 mL of absolute ethanol, add 30 mL of pure water, mix gently by pipetting, and store frozen at -20°C. This embodiment includes a cell membrane-breaking solution with the following formula:

Sodium dihydrogen phosphate 0.12%;

Disodium hydrogen phosphate 0.14%;

Sodium chloride 0.88%;

Triton X-100 0.2%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at room temperature.

Example 3

The apoptotic ratio of HL-60 cells was detected using the fixed membrane-breaking protocols of Example 1 and Example 2 respectively.

Take the logarithmically growing HL-60 cells, centrifuge at 300g for 5 minutes, discard the supernatant; resuspend the cell pellet in IMDM complete medium, count the cells, adjust the cell density to 1×10 ⁶ /mL, inoculate a 6-well plate, and add a final concentration of 2.5 μM camptothecin, mix by gently pipetting, induce and culture in 37°C CO ₂ incubator for 3 hours, collect the cell pellet, centrifuge at 300g for 5 minutes, discard the supernatant; add 1× ¹⁰ cells per group, add 100 μL PBS to resuspend Cells are precipitated, then add an equal volume of fixative, pipet gently to mix, and fix at room temperature for 60 minutes; add 1mL PBS buffer, pipet gently to mix, centrifuge at 300g for 5 minutes, discard the supernatant; add 100 μL membrane-breaking solution, and resuspend the cells Precipitate, rupture the membrane at room temperature for 30 minutes, add PBS (1% BSA) to the maximum volume to terminate the membrane rupture, centrifuge at 300g for 5 minutes, discard the supernatant; add 100 μL balance solution to each group to balance at 37°C for 15 minutes, centrifuge at 300g for 5 minutes, discard the supernatant; press The cells after membrane rupture were stained by TUNEL method and tested on the machine.

The results are shown in Figure 1. It can be seen that compared with unfixed living cells, the fixed membrane rupture scheme provided in Example 1 and the fixed membrane rupture scheme provided in Example 2 both resulted in significantly reduced cell morphology, lower FSC value, and lower cell loss. The yield is low, and no apoptosis signal is detected, so it is not suitable for the detection of TUNEL-labeled DNA.

Example 4

This embodiment includes a cell fixative with the following formula:

Paraformaldehyde 0.5%

Methanol 10%;

Measure 10 mL of 100% methanol, weigh 0.5 g of paraformaldehyde, dissolve it in 100 mL of pure water, and store in a freezer at -20°C away from light.

This embodiment includes a cell membrane-breaking solution with the following formula:

Sodium dihydrogen phosphate 0.12%;

Disodium hydrogen phosphate 0.14%;

Sodium chloride 0.88%;

Triton X-100 0.2%;

Fetal bovine serum 3%

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at room temperature.

Example 5

This embodiment includes a cell fixative with the following formula:

37~40% formaldehyde solution 10%;

Sodium dihydrogen phosphate 0.12%;

Disodium hydrogen phosphate 0.14%;

Sodium chloride 0.88%;

Potassium chloride 0.02M;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at 4°C.

This embodiment includes a cell membrane-breaking solution with the following formula:

Sodium dihydrogen phosphate 0.12%;

Disodium hydrogen phosphate 0.14%;

Sodium chloride 0.88%;

Potassium chloride 0.15%;

Triton X-100 0.2%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at 4°C.

Example 6

This example uses the fixed membrane-breaking protocol of Example 2, Example 4, and Example 5 to verify the apoptosis results of mouse spleen cells.

Prepare mouse spleen single cell suspension, centrifuge at 300g for 5 minutes, discard the supernatant; add 2 mL of freshly prepared 1X ACK red blood cell lysis solution, lyse at room temperature for 2 minutes, add 10 mL of PBS buffer to stop lysis, centrifuge at 300g for 5 minutes, discard the supernatant; 1640 Resuspend the cell pellet in the full culture medium, count the cells, adjust the cell density to 1×10 ⁶ /mL, and inoculate a 6-well plate. Add camptothecin with a final concentration of 2.5 μM to the induction group, mix by gently pipetting, and incubate at 37°C CO ₂ After induction culture in the incubator for 3 hours, collect the cell pellet, centrifuge at 300g for 5 minutes, discard the supernatant; add 1×10 ⁶ cells per group, add 100 μL PBS to resuspend the cell pellet, then add an equal volume of fixative, and mix gently by pipetting. Fix at room temperature in the dark for 60 minutes; add 1 mL of PBS buffer, mix by gently pipetting, centrifuge at 300g for 5 minutes, discard the supernatant; add 100 μL of rupture solution, resuspend the cell pellet, rupture the membrane on ice for 10 minutes, and add PBS (1% BSA ) to the maximum volume to terminate the membrane rupture, centrifuge at 300g for 5 minutes, discard the supernatant; stain the cells after membrane rupture according to the TUNEL method, and use the machine for detection.

The results are shown in Figure 2. It can be seen that the fixation effect of formaldehyde solution is better than that of glacial ethanol and glacial methanol, but the detection effect of negative and positive groups of apoptotic cells is poor; the fixation and membrane rupture scheme of glacial ethanol and glacial methanol resulted in The sample is false positive (the results are all positive).

Example 7

This embodiment includes a cell fixative with the following formula: 37-40% formaldehyde solution 5%;

Methanol 5%;

Sodium dihydrogen phosphate 0.12%;

Disodium hydrogen phosphate 0.14%;

Sodium chloride 0.88%;

Potassium chloride 0.15%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at 4°C. This embodiment includes a cell membrane-breaking solution with the following formula:

Sodium dihydrogen phosphate 0.12%;

Disodium hydrogen phosphate 0.14%;

Sodium chloride 0.88%;

Potassium chloride 0.15%;

Triton X-100 0.2%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at 4°C.

Example 8

This embodiment includes a cell fixative with the following formula: 37-40% formaldehyde solution 5%;

Glutaraldehyde 2%;

Methanol 5%;

Sodium dihydrogen phosphate 0.12%;

Disodium hydrogen phosphate 0.14%;

Sodium chloride 0.88%;

Potassium chloride 0.15%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at -20°C. This embodiment includes a cell membrane-breaking solution with the following formula:

Sodium dihydrogen phosphate 0.12%;

Disodium hydrogen phosphate 0.14%;

Sodium chloride 0.88%;

Potassium chloride 0.15%;

Triton X-100 0.2%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at 4°C.

Example 9

This embodiment includes a cell fixative with the following formula:

Glutaraldehyde 2%;

Paraformaldehyde 8%;

Methanol 10%;

Triton X-100 0.3%;

Sodium chloride 5%;

Potassium chloride 0.2%;

Potassium dihydrogen phosphate 2%;

Disodium hydrogen phosphate dodecahydrate 0.5%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at -20°C.

This embodiment includes a cell membrane-breaking solution with the following formula:

NP40 1%;

Triton X-100 0.2%;

Sodium chloride 5%;

Potassium chloride 0.2%;

Potassium dihydrogen phosphate 2%;

Trisodium citrate dihydrate 3%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at 4°C.

Example 10

This example uses the fixed membrane-breaking protocols of Examples 7, 8 and 9 to detect the apoptosis ratio of Jurkat cells.

Collect Jurkat cells in the logarithmic growth phase, centrifuge at 300g for 5 minutes, discard the supernatant; resuspend the cell pellet in 1640 complete medium, count the cells, adjust the cell density to 1×10 ⁶ /mL, inoculate into a 6-well plate, and add a final concentration of 5 μM of camptothecin, mix gently by pipetting, induce and culture in a 37°C CO ₂ incubator for 3 hours, collect the cell pellet, centrifuge at 300g for 5 minutes, discard the supernatant; add 1× ¹⁰ cells per group, add 100 μL PBS to resuspend the cell pellet , then add an equal volume of fixative, pipet gently to mix, and fix at room temperature for 30 minutes; add 1mL PBS buffer, pipet gently to mix, centrifuge at 300g for 5 minutes, discard the supernatant; add 100 μL membrane-breaking solution, resuspend the cell pellet, The membrane was ruptured for 30 minutes at room temperature, and PBS (1% BSA) was added to the maximum volume to terminate the membrane rupture. Centrifuge at 300g for 5 minutes and discard the supernatant. The cells after membrane rupture were stained according to the TUNEL method and tested on the machine.

The results are shown in Figure 3. It can be seen that the ratio of formaldehyde and paraformaldehyde in the fixative solution cannot improve the cell yield in the final product. Adding glutaraldehyde is beneficial to increasing the cell yield and the cell status in the FSC chart; cell debris More, membrane rupture conditions need to be optimized.

Example 11

This example uses the fixed membrane rupture schemes of Examples 7, 8 and 9 to optimize membrane rupture conditions.

Collect Jurkat cells in the logarithmic growth phase, centrifuge at 300g for 5 minutes, discard the supernatant; resuspend the cell pellet in 1640 complete medium, count the cells, adjust the cell density to 1×10 ⁶ /mL, inoculate into a 6-well plate, and add a final concentration of 5 μM of camptothecin, mix gently by pipetting, induce and culture in a 37°C CO ₂ incubator for 3 hours, collect the cell pellet, centrifuge at 300g for 5 minutes, discard the supernatant; add 1× ¹⁰ cells per group, add 100 μL PBS to resuspend the cell pellet , then add an equal volume of fixative, mix by gently pipetting, and fix at room temperature for 60 minutes; add 1 mL of PBS buffer, mix by gently pipetting, centrifuge at 300g for 5 minutes, and discard the supernatant; add 100 μL of membrane-breaking solution, and resuspend the cell pellet. The membrane was ruptured on ice for 10 minutes, and PBS (1% BSA) was added to the maximum volume to terminate the membrane rupture. Centrifuge at 300g for 5 minutes, and the supernatant was discarded. The cells after membrane rupture were stained according to the TUNEL method and tested on the machine.

The results are shown in Figure 4. It can be seen that extending the fixation time to 60 minutes is beneficial to improving cell yield and maintaining cell morphology; optimizing the membrane rupture time to 10 minutes on ice is beneficial to reducing cell debris, but it still cannot effectively break the nuclear membrane.

Example 12

This embodiment includes a cell fixative with the following formula:

Glutaraldehyde 0.1%;

Paraformaldehyde 1%;

Methanol 0.5%;

Glycerin 0.5%;

Triton X-1000.5%;

Sodium chloride 0.1%;

Potassium chloride 0.1%;

Potassium dihydrogen phosphate 0.1%;

Sodium hydrogen phosphate dodecahydrate 2%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at -20°C.

This embodiment includes a cell membrane-breaking solution with the following formula:

NP40 0.02%;

Triton X-100 0.01%;

BSA 0.2%;

Sodium chloride 0.1%;

Potassium chloride 0.1%;

Potassium dihydrogen phosphate 0.1%;

Trisodium citrate dihydrate 2%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at 4°C.

Example 13

This example uses the fixed membrane rupture protocol in Example 12 to detect the apoptosis ratio of Jurkat cells.

Collect Jurkat cells in the logarithmic growth phase, centrifuge at 300g for 5 minutes, discard the supernatant; resuspend the cell pellet in 1640 complete medium, count the cells, adjust the cell density to 1×10 ⁶ /mL, inoculate into a 6-well plate, and add a final concentration of 5 μM of camptothecin, mix gently by pipetting, induce and culture in a 37°C CO ₂ incubator for 3 hours, collect the cell pellet, centrifuge at 300g for 5 minutes, discard the supernatant; add 1× ¹⁰ cells per group, add 100 μL PBS to resuspend the cell pellet , then add an equal volume of fixative, mix by gently pipetting, and fix at room temperature for 60 minutes; add 1 mL of PBS buffer, mix by gently pipetting, centrifuge at 300g for 5 minutes, and discard the supernatant; add 100 μL of membrane-breaking solution, and resuspend the cell pellet. The membrane was ruptured on ice for 10 minutes, and PBS (1% BSA) was added to the maximum volume to terminate the membrane rupture. Centrifuge at 300g for 5 minutes, and the supernatant was discarded. The cells after membrane rupture were stained according to the TUNEL method and tested on the machine.

The results are shown in Figure 5. It can be seen that increasing the membrane-breaking components in the fixative and extending the fixation time to 60 minutes will help improve the cell yield and maintain cell morphology; optimize the membrane-breaking components and add the protective effect of BSA, adjust The membrane rupture time is 5 minutes on ice, which is beneficial to reducing cell debris and can effectively break the nuclear membrane, but the grouping effect is slightly poor.

On the basis of Example 13, the membrane rupture time was adjusted to 20 min and 30 min respectively.

The results are shown in Figure 6. It can be seen that maintaining the fixation time of the fixative to 60 minutes is beneficial to improving cell yield and maintaining cell morphology; optimizing the composition of the membrane rupture solution, adding the protective effect of BSA, and adjusting the membrane rupture time to on ice. 10, 20, and 30 minutes can effectively break the nuclear membrane. From the analysis of the proportion of positive groups and average fluorescence brightness, the membrane breaking time of 10 minutes on ice has the best effect.

Example 14

This embodiment includes a cell fixative with the following formula:

Glutaraldehyde 5%;

Paraformaldehyde 10%;

Methanol 5%;

Glycerin 5%;

Triton X-1002%;

Sodium chloride 1%;

Potassium chloride 0.5%;

Potassium dihydrogen phosphate 0.5%;

Disodium hydrogen phosphate dodecahydrate 0.5%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at -20°C.

This embodiment includes a cell membrane-breaking solution with the following formula:

NP40 1%;

Triton X-100 3%;

BSA5%;

Sodium chloride 0.5%;

Potassium chloride 0.5%;

Potassium dihydrogen phosphate 0.5%;

Trisodium citrate dihydrate 0.5%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at 4°C.

Example 15

This example uses the fixed membrane rupture protocol in Example 14 to detect the apoptosis ratio of HL-60 cells and Jurkat cells.

Collect Jurkat cells and HL-60 cells in the logarithmic growth phase, centrifuge at 300g for 5 minutes, discard the supernatant; resuspend the cell pellet in 1640 complete medium, count the cells, adjust the cell density to 1×10 ⁶ /mL, and inoculate into a 6-well plate. Add camptothecin at a final concentration of 5 μM to Jurkat cells (camptothecin to a final concentration of 2.5 μM for HL-60 cells), mix by gently pipetting, and induce and culture in a 37°C CO ₂ incubator for 3 hours. Collect the cell pellet, 300g. Centrifuge for 5 minutes, discard the supernatant; add 1×10 ⁶ cells in each group, add 100 μL PBS to resuspend the cell pellet, then add an equal volume of fixative, mix gently by pipetting, and fix at room temperature for 60 minutes; add 1mL PBS buffer and pipet gently Mix well, centrifuge at 300g for 5 minutes, discard the supernatant; add 100 μL of membrane rupture solution, resuspend the cell pellet, rupture the membrane on ice for 10 minutes, add PBS (1% BSA) to the maximum volume to terminate the membrane, centrifuge at 300g for 5 minutes, discard the supernatant ; Stain the cells after membrane rupture according to the TUNEL method and test on the machine.

The results are shown in Figure 7. Adjust the ratio of each reagent within a certain range, maintain the fixation time of the fixative to room temperature for 60 minutes, and adjust the membrane rupture time to 10 minutes on ice. The nuclear membrane can be effectively broken, and Jurkat cells and HL-60 can be detected. The proportion of cells undergoing apoptosis.

Example 16

This embodiment includes a cell fixative with the following formula:

Glutaraldehyde 1%;

Paraformaldehyde 25%;

Methanol 10%;

Glycerin 10%;

Triton X-1003%;

Sodium chloride 3%;

Potassium chloride 0.2%;

Potassium dihydrogen phosphate 0.2%;

Disodium hydrogen phosphate dodecahydrate 0.2%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at -20°C.

This embodiment includes a cell membrane-breaking solution with the following formula:

NP40 3%;

Triton X-100 0.1%;

BSA10%;

Sodium chloride 3%;

Potassium chloride 0.2%;

Potassium dihydrogen phosphate 0.2%;

Trisodium citrate dihydrate 0.2%;

Accurately weigh each of the above reagents, dissolve and mix thoroughly, adjust the pH to 7.2, and store in the dark at 4°C.

This example uses the optimized fixed membrane-breaking protocol in Example 16 to detect the apoptosis ratio of adherent HeLa cells.

Digest HeLa cells with trypsin, centrifuge at 300g for 5 minutes, discard the supernatant; resuspend the cell pellet in 1640 complete medium, count the cells, adjust the cell density to 2×10 ⁵ /mL, inoculate into a 6-well plate, and culture in a 37°C CO ₂ incubator overnight , when the cell density reaches 90%, add camptothecin with a final concentration of 2.5 μM to the induction group, mix gently by pipetting, and after induction and culture in a 37°C CO ₂ incubator for 6 hours, collect the supernatant of each group and trypsinize the Hela cells at the same time. , collect the cell pellet, centrifuge at 300g for 5 minutes, discard the supernatant; add 1×10 ⁶ cells per group, add 100 μL PBS to resuspend the cell pellet, then add an equal volume of fixative, mix gently by pipetting, and fix at room temperature for 60 min; add 1mL PBS buffer, mix by gently pipetting, centrifuge at 300g for 5 minutes, discard the supernatant; add 100 μL membrane rupture solution, resuspend the cell pellet, rupture the membrane on ice for 10 minutes, add PBS (1% BSA) to the maximum volume to terminate the membrane rupture , centrifuge at 300g for 5 minutes, discard the supernatant; stain the cells after membrane rupture according to the TUNEL method, and test on the machine.

The results are shown in Figure 8. By adjusting the ratio of each reagent within a certain range, maintaining the fixation time of the fixative to room temperature for 60 minutes, and adjusting the membrane rupture time to 10 minutes on ice, the apoptosis ratio of adherent HeLa cells can be effectively detected.

Example 17

This example uses the fixed membrane rupture protocol in Example 16 to detect the apoptosis ratio of mouse spleen primary cells.

Prepare mouse spleen single cell suspension, centrifuge at 300g for 5 minutes, discard the supernatant; add 2 mL of freshly prepared 1X ACK red blood cell lysis solution, lyse at room temperature for 2 minutes, add 10 mL of PBS buffer to stop lysis, centrifuge at 300g for 5 minutes, discard the supernatant; 1640 Resuspend the cell pellet in the full culture medium, count the cells, adjust the cell density to 1×10 ⁶ /mL, and inoculate a 6-well plate. Add camptothecin with a final concentration of 2.5 μM to the induction group, mix by gently pipetting, and incubate at 37°C CO ₂ After induction and culture in the incubator for 3 hours, collect the cell pellet, centrifuge at 300g for 5 minutes, discard the supernatant; add 1×10 ⁶ cells per group, add 100 μL PBS to resuspend the cell pellet, add CD3 phenotype antibody, and incubate at 4°C for 30 min; add PBS (1% BSA) to the maximum volume to terminate membrane rupture, centrifuge at 300g for 5 minutes, discard the supernatant; add 100 μL PBS to resuspend the cell pellet, then add an equal volume of fixative, mix gently by pipetting, and fix at room temperature for 60 minutes in the dark; add 1 mL PBS buffer, mix gently by pipetting, centrifuge at 300g for 5 minutes, discard the supernatant; add 100 μL of membrane rupture solution, resuspend the cell pellet, rupture the membrane on ice for 10 minutes, add PBS (1% BSA) to the maximum volume to terminate the membrane rupture. Centrifuge at 300g for 5 minutes and discard the supernatant; stain the cells after membrane rupture according to the TUNEL method and run the test machine for detection.

The results are shown in Figure 9: Maintaining the fixation time of the fixative to room temperature for 60 minutes, and adjusting the membrane rupture time to 10 minutes on ice, the apoptosis ratio in the primary cell mouse spleen single cell suspension can be effectively detected, and can Co-staining with other phenotypic staining antibodies does not affect the staining results.

Example 18

This example uses the optimized fixed membrane-breaking protocol in Example 16 to detect the apoptosis ratio of Jurkat labeled with different fluorescently labeled staining solutions.

Collect Jurkat cells in the logarithmic growth phase, centrifuge at 300g for 5 minutes, discard the supernatant; resuspend the cell pellet in 1640 complete medium, count the cells, adjust the cell density to 1×10 ⁶ /mL, inoculate a 6-well plate, and add Jurkat cells to the final Camptothecin at a concentration of 5 μM was mixed by gently pipetting. After induction and culture in a 37°C _CO2 incubator for 3 hours, the cell pellet was collected, centrifuged at 300 g for 5 min, and the supernatant was discarded; each group had 1 × ¹⁰ cells, and 100 μL PBS was added to rehydrate. Suspend the cell pellet, then add an equal volume of fixative, mix gently by pipetting, and fix at room temperature for 60 minutes; add 1 mL PBS buffer, mix gently by pipetting, centrifuge at 300g for 5 minutes, discard the supernatant; add 100 μL membrane-breaking solution, and resuspend Cells were precipitated, and the membrane was ruptured on ice for 10 minutes. PBS (1% BSA) was added to the maximum volume to terminate the membrane rupture. Centrifuge at 300g for 5 minutes, and the supernatant was discarded. The cells after membrane rupture were stained according to the TUNEL method and tested on the machine.

The results are shown in Figure 10. It can be seen that by maintaining the fixation time of the fixative at room temperature for 60 minutes and adjusting the membrane rupture time to 10 minutes on ice, labeling solutions with different fluorescence channels can be detected, and the results of the detected cell apoptosis ratio are consistent.

In summary, conventional flow cytometry fixation rupture solution can effectively break the outer cell membrane, but it cannot further break the nuclear membrane and ensure effective labeling of nuclear DNA staining. This technology adopts an effective fixed membrane-breaking solution. In the cell samples tested by TUNEL flow cytometry, the fixed membrane-breaking solution can fix the cells overnight at 4°C or fix the cells at room temperature for 1 hour, and rupture the membrane on ice for 5 to 20 minutes. It can both maintain The cells are in good condition and can permeate the cell outer membrane while effectively punching holes in the cell nucleus, allowing the TDT enzyme and labeling solution components in the TUNEL flow cytometry experiment to smoothly enter the cell nucleus to complete effective labeling of broken DNA. Improved the success rate, repeatability, sensitivity and resolution of TUNEL flow detection.

The above-described embodiments and features in the embodiments herein may be combined with each other if there is no conflict.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (10)

1. A TUNEL special fixed membrane-breaking agent for flow cytometric analysis of apoptotic cells, characterized in that the fixed membrane-breaking liquid includes a fixative and a membrane-breaking liquid; in terms of weight percentage, the fixed membrane includes Glutaraldehyde 0.1 to 5%, paraformaldehyde 1% to 25%, methanol 0.5% to 10%, glycerin 0.5% to 10%, Triton X-100 0.5% to 5%, sodium chloride 0.1% to 3%, Potassium chloride 0.1% ~ 1%, potassium dihydrogen phosphate 0.1% ~ 1%, sodium hydrogen phosphate dodecahydrate 0.1% ~ 2% and the balance is water; in terms of weight percentage, the membrane-breaking solution includes NP40 0.02 %～3%, Triton And trisodium citrate dihydrate 0.1% to 2% and the balance is water. 2. The fixed membrane-breaking agent according to claim 1, wherein the pH of the fixing solution is 7-7.6, and the pH of the membrane-breaking solution is 7-7.6. 3. A method for preparing a fixative as claimed in claim 1 or 2, characterized in that it includes the following steps: dissolving paraformaldehyde in pure water, and then weighing and adding sodium chloride and potassium chloride in sequence. , potassium dihydrogen phosphate and disodium hydrogen phosphate dodecahydrate, stir and dissolve at room temperature, adjust the pH to 7.0~7.6, then add methanol solution, stir while adding, wait until the solution is mixed, then add glutaraldehyde liquid, glycerin and Triton X-100, stir and mix at room temperature, let it stand to eliminate bubbles and then dilute to volume to obtain the fixative. 4. a kind of preparation method of membrane-breaking liquid as claimed in claim 1 or 2, is characterized in that, comprises the following steps: sequentially weigh sodium chloride, potassium chloride, potassium dihydrogen phosphate and trisodium citrate dihydrate , add to pure water, stir and mix at room temperature, adjust the pH to 7.0~7.6, weigh and add NP40, stir and mix, add Triton , stir and mix at room temperature, adjust the volume to 1L, aliquot and store at -20°C to obtain the membrane-breaking solution. 5. Application of the TUNEL-specific fixed membrane-breaking agent according to claim 1 or 2 in detecting apoptosis of cell samples by TUNEL method. 6. The application according to claim 5, wherein the cell sample includes any one of suspension cells, single cell suspension, adherent cells and primary cells. 7. The application according to claim 5, characterized in that it is used to break the nuclear membrane of the cell nucleus and stain the DNA in the cell nucleus. 8. A method for detecting cell apoptosis in flow cytometry using the TUNEL-specific fixed membrane-breaking agent as claimed in claim 1 or 2, which is characterized in that it includes the following specific steps: S1. Collect the cell sample, add PBS buffer to resuspend the cell pellet, and then add the fixative for fixation; S2. Centrifuge the fixed cells, discard the supernatant, add the membrane rupture solution, resuspend the cell pellet for membrane rupture, terminate the membrane rupture, centrifuge again, and discard the supernatant; S3. Stain the cells after membrane rupture according to the TUNEL method and use the machine for detection. 9. The method of claim 8, wherein the number of collected cell samples is 1 to 5× ¹⁰ per detection tube, and the cell pellet is resuspended in 100 to 600 μL of PBS and mixed evenly. The volume of added fixative solution is 100-600 μL, the fixation time is 45-60 minutes, and the fixation condition is room temperature. 10. The method of claim 8, wherein 100 to 600 μL of membrane-breaking solution is added to the centrifuged cells, and the cell pellet is resuspended and mixed. The membrane-breaking conditions are 5 to 20 minutes on ice. After the membrane, 1 to 2 mL of PBS buffer needs to be added to terminate the membrane rupture. The PBS buffer contains 1% BSA.