CN106868148B - Preparation method of cell nucleus with controllable aggregation state and long-term preservation - Google Patents
Preparation method of cell nucleus with controllable aggregation state and long-term preservation Download PDFInfo
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Abstract
The scheme relates to a preparation method of cell nucleuses with controllable aggregation states and long-term preservation, which comprises the following steps: collecting a nucleated cell sample, washing, centrifuging to remove a supernatant, and adding a lysis solution for lysis; adding sucrose solution, and centrifuging at 15000-; taking the supernatant, adding the obtained cell nucleus into a pre-cooled stationary liquid 1 with the temperature of-20 ℃ for crosslinking; after crosslinking for 2 days, the fixative 1 is replaced by fixative 2, and the cell nucleus with controllable aggregation state and long storage life is obtained. The preparation method of the cell nucleus in the scheme can be completed by only 3 steps and can be used as a good product development scheme; the dependence of the domestic flow cytometer on imported reagents is gradually changed, and the application cost is greatly reduced; meanwhile, the mass production of the reagent can promote the cell ploidy detection and clinical tumor prognosis in scientific research, and improve the tumor treatment quality. The red cell nucleus product obtained by the scheme can be stably stored for more than 1 year at the temperature of 4 ℃, and has good product development potential.
Description
Technical Field
The invention belongs to the field of reagents for medical detection, and particularly relates to a preparation method of a cell nucleus with controllable aggregation state and long-term preservation.
Background
The flow cytometer is one of the most advanced apparatuses in the fields of life science and clinical cell cycle and apoptosis detection, tumor detection and prognosis, etc., and can simultaneously and high-flux detect cell DNA information so as to perform detailed analysis on the cell cycle, apoptosis state and ploidy condition. In performing the above-described detection process, it is often necessary to calibrate the accuracy of the instrument. The cell nucleus is used as the main storage place of genetic material in the cell, has stable and consistent DNA content, can form 1,2,3, 4 to more than 7 cell nucleus aggregation groups after special treatment, can calibrate the linearity, the detection limit and the amplification gain of a flow cytometer after being dyed by fluorescent dye, and can be used as an internal standard product in DNA detection. The developed cell nucleus quality control reagents comprise chicken erythrocyte nuclei, calf thymine nuclei, trout erythrocyte nuclei and the like, the cost of the cell nucleus reagents is lower than that of chemically synthesized microspheres, the cell nucleus reagents have the same composition with biological samples, and the cell nucleus reagents can be better applied to calibration of flow DNA detection.
However, there is no development scheme for a cell nucleus calibration reagent disclosed at home and abroad currently, and a cell nucleus reagent for calibrating a flow cytometer in the market is mainly monopolized by BD and Biosure companies, is expensive, has a long shelf life, and causes much inconvenience for the application of the flow cytometer.
Disclosure of Invention
In view of the disadvantages of the prior art, the object of the present invention is to provide a standard method for preparing cell nuclei with controllable aggregation state and long storage life.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a method for producing a cell nucleus with a controlled aggregation state and capable of long-term storage, which comprises:
s1, collecting a nucleated cell sample, washing, centrifuging to remove a supernatant, and adding a lysis solution for lysis;
s2 adding sucrose solution, centrifuging at 15000-40000g and 4 ℃;
s3, taking the supernatant, adding the obtained cell nucleus into a pre-cooled stationary liquid 1 at the temperature of-20 ℃ for crosslinking;
s4, after crosslinking for 2 days, replacing the stationary liquid 1 with the stationary liquid 2 to obtain the cell nucleus with controllable aggregation state and long-term preservation;
wherein the fixing liquid 1 includes:
methanol or ethanol;
bovine serum albumin or fetal bovine serum or glycerol;
water;
the fixing liquid 2 includes:
glutaraldehyde or formaldehyde;
bovine serum albumin or fetal bovine serum or glycerol;
and (3) water.
Preferably, the cell nucleus preparation method with controllable aggregation state and long-term preservation is adopted, wherein the lysate comprises 0.1-0.5% of triton X-100, 0.1-2 μ M of dithiothreitol, 0.1-1 μ M of phenylmethylsulfonyl fluoride, 0.5-2% of ethylphenylpolyethylene glycol, 30-60mM of sodium citrate or tris (hydroxymethyl) aminomethane, 0.01-0.1% (v/v) of DMSO and pH 7.6.
The related cells may include chicken erythrocytes, calf thymine cells, zebra fish erythrocytes, trout erythrocytes and the like, most of the cells are nucleated erythrocytes or other cells with wide sources and nucleated cells, the concentration and the time are preferred, the cell membranes are broken and the nuclear membranes are complete, wherein the triton is a cell permeation agent, the dithiothreitol is a sulfhydryl protective agent, the phenylmethylsulfonyl fluoride is a protease inhibitor, the ethylphenylpolyethylene glycol is cell lysate, the sodium citrate and the tris (hydroxymethyl) aminomethane have the buffering effect, more importantly, the breaking effect of the lysate on the nuclear membranes is relieved, and the DMSO is a nuclear membrane flow protective agent.
Preferably, the method for preparing cell nuclei with controllable aggregation state and long preservation time comprises the steps of dissolving 1.5-2.2M of sucrose, 0.5-2% of glycerol and 0.1-0.5% of glycine in 0.01M of PBS buffer solution in the sucrose solution.
The concentration of the sucrose solution and the centrifugal speed are preferably selected, the separation of cell nucleus and cytoplasmic membrane components is ensured, the integrity of the nuclear membrane and DNA in the nuclear membrane is protected in the process, the glycerol is a nuclear membrane stabilizer, the glycine is a newly introduced reagent, and the protection effect of the glycine on the nuclear membrane stability is mainly considered.
Preferably, the preparation method of cell nucleus with controllable aggregation state and long-term preservation is characterized in that the fixative 1 contains 70-80% of methanol or ethanol, 1-10% of bovine serum albumin or fetal bovine serum or glycerol, and the balance is filled with water.
Wherein methanol and ethanol are used for fixing chromatin in cell nucleus, protecting DNA in the chromatin, and realizing aggregation of cell nucleus. Serum, protein or glycerol prevent nuclear membrane disruption during concussion. The proportion of the ethanol and the protective agent is optimized, so that the controllable crosslinking of the chicken erythrocyte nucleus is realized, and the requirements of 30-40% of mononuclear, 15-20% of binuclear, 5-15% of trinuclear, 5-10% of tetranuclear, 2-10% of quinucleus, 1-5% of hexanuclear and 1-5% of heptanuclear are met, so that the requirements are met.
Preferably, the preparation method of the cell nucleus with controllable aggregation state and long-term preservation is adopted, wherein the fixing solution 2 contains 2% of glutaraldehyde or 1% of formaldehyde, 1-10% of bovine serum albumin or fetal bovine serum or glycerol, and the balance is filled with water, wherein the glutaraldehyde or the formaldehyde is mainly used for maintaining the aggregation state of the cell nucleus.
Preferably, the preparation method of the cell nucleus with controllable aggregation state and long-term preservation capability is characterized in that the stationary liquid 1 further comprises 0.3-0.5% of furfural, which is a good organic solvent and has active chemical properties, and a plurality of derivatives can be prepared through reactions such as oxidation, condensation and the like, so that the preparation method has strong bactericidal capability.
Preferably, the preparation method of the cell nucleus with controllable aggregation state and long-term preservation capability is characterized in that the stationary liquid 1 further comprises 0.05-0.08% of ethyl maltol, which is a safe and nontoxic additive with wide application, good effect and extremely small dosage, and has antibacterial and antiseptic properties and can prolong the storage period.
The invention has the beneficial effects that:
1. the reagent can be used as a calibrator and a quality control product in the total amount detection, the period analysis and the tumor prognosis detection of cell DNA of a flow cytometer and related instruments.
2. Gradually changing the dependence of the reagent for the flow cytometer in China on imported reagents and greatly reducing the application cost of the flow cytometer.
3. The mass production of the reagent can promote the convenience of cell ploidy detection and clinical tumor prognosis in scientific research, and improve the tumor treatment quality.
4. The preparation method of the cell nucleus in the scheme can be completed by only 3 steps, and the obtained reagent has high purity and the same quality as an imported product, and can meet the requirements of instruments.
5. The reagents required by the method are low in price, the preparation cost of the reagents can be effectively reduced, and the method can be well used as a product development scheme.
6. The red cell nucleus product obtained by the scheme can be stably stored for more than 1 year at the temperature of 4 ℃, and has good product development potential.
Drawings
FIG. 1 is a graph showing the effect of a chicken red blood cell nuclear reagent (embodiment 1) on flow cytometry obtained by a method for producing a cell nucleus with controllable aggregation state and capable of long-term preservation.
FIG. 2 is a 200-fold microscopic image of a chicken red blood cell nucleus (embodiment 1) obtained by the method for producing a nucleus capable of controlling the aggregation state and long-term preservation.
FIG. 3 is a graph showing the effect of detecting calf thymine nucleus reagent (embodiment 2) on a flow cytometer using a method for preparing nuclei having a controlled aggregation state and capable of long-term preservation.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
Six embodiments of a method for preparing cell nuclei with controlled aggregation and long-term preservation and two comparative embodiments are listed below, including:
(1) Collection 3 x 107The chicken red blood cells were washed with PBS, centrifuged at 1500rpm to remove the supernatant, and 10mL of a lysate containing 0.1% Triton X-100, 1. mu.M dithiothreitol and 0.5. mu.M phenylmethylsulfonyl fluoride, 1% ethylphenylpolyethylene glycol, 40mM sodium citrate, 0.05% DMSO, pH7.6 was added.
(2) After 10min of lysis, the chicken red blood cell solution was slowly added to the upper layer of 30mL concentrated sucrose solution, centrifuged at 30,000 g and 4 ℃ for 50 min. The sucrose solution contained 2M sucrose, 1% glycerol, 0.2% glycine dissolved in 0.01M PBS buffer.
(3) And (3) adding a fixing solution precooled at the temperature of-20 ℃ into the obtained chicken erythrocyte nuclei, wherein the fixing solution contains 40% of methanol, 30% of ethanol, 1% of glutaraldehyde, 1% of formaldehyde, 1% of bovine serum albumin, 1% of glycerol and the balance of water. After fixation, the cells showed different levels of multimeric aggregate structures (1,2,3-7 nuclear aggregates, FIG. 1).
All the processes should be kept at 4 ℃. The obtained chicken red blood cell nucleus is stained by 5 mu g/ml propidium iodide, and detected by a flow cytometer to form 7 peaks, the proportion meets the requirement, the single-peak CV value is between 2.3% and 2.8% respectively (figure 2), and the method can be used for subsequent calibration tests.
In the scheme, the cells in the embodiment 1 are replaced by calf thymine cells, the rest cells are kept unchanged, the obtained calf thymine cell nucleus is stained by 5 mu g/ml propidium iodide, the 7 peaks are detected by a flow cytometer, the ratio meets the requirement, the single-peak CV values are respectively between 1.8% and 3.0% (figure 3), and the requirement of a subsequent calibration test can also be met.
Embodiment 3
The cell in the embodiment 1 is replaced by the zebra fish erythrocyte, the rest of the zebra fish erythrocyte is kept unchanged, the obtained zebra fish erythrocyte nucleus is stained by 5 mu g/ml propidium iodide, the zebra fish erythrocyte nucleus is detected by a flow cytometer to form 8 peaks, the proportion meets the requirement, the single-peak CV values are respectively between 2.1% and 2.9%, and the requirement of a subsequent calibration test can be met.
Embodiment 4
The cell in the embodiment 1 is replaced by the trout erythrocyte, the rest is kept unchanged, the cell nucleus of the trout erythrocyte is stained by 5 mu g/ml propidium iodide, the cell nucleus is detected by a flow cytometer to form 7 peaks, the proportion meets the requirement, the single-peak CV value is respectively between 2.5% and 2.9%, and the requirement of a subsequent calibration test can be met.
Embodiment 5
In the scheme, 0.3-0.5% of furfural is added into the stationary liquid 1 in the embodiment 1, the rest furfural is kept unchanged, the obtained chicken red blood cell nucleus is stained by 5 mu g/ml propidium iodide, and the peak is detected by a flow cytometer to form 8 peaks, the proportion meets the requirement, the single-peak CV value is respectively 1.5% -1.7%, and the requirement of a subsequent calibration test can be met.
Embodiment 6
In the scheme, 0.05-0.08% of ethyl maltol is added into the stationary liquid 1 in the embodiment 5, the rest is kept unchanged, the obtained chicken red blood cell nucleus is stained by 5 mu g/ml of propidium iodide, and the chicken red blood cell nucleus becomes 9 peaks through detection of a flow cytometer, the proportion meets the requirement, the single-peak CV values are respectively 1.3-1.4%, and the requirement of a subsequent calibration test can be met.
0.05-0.08% of ethyl maltol is added into the fixing solution 1 in the embodiment 1, furfural is not added, the rest is kept unchanged, the obtained chicken red blood cell nucleus is stained by 5 mu g/ml of propidium iodide, and the peak is formed by detection of a flow cytometer, the proportion is not in accordance with requirements, and the single-peak CV value is respectively between 3.3-3.6%, so that the requirements of subsequent calibration tests are not met.
0.2% of furfural and 0.05-0.08% of ethyl maltol are added into the stationary liquid 1 in the embodiment 1, the rest is kept unchanged, the obtained chicken red blood cell nucleus is stained by 5 mu g/ml of propidium iodide, and the peak becomes 7 by detection of a flow cytometer, the proportion does not meet the requirement, and the single-peak CV values are respectively between 2.4-2.9%, so that the requirement of a subsequent calibration test is not met.
Comparative scheme 3
0.6% of furfural and 0.05-0.08% of ethyl maltol are added into the stationary liquid 1 in the embodiment 1, the rest is kept unchanged, the obtained chicken red blood cell nucleus is stained by 5 mu g/ml of propidium iodide, and the peak becomes 7 by detection of a flow cytometer, the proportion does not meet the requirement, and the single-peak CV values are respectively between 2.5-2.8%, so that the requirement of a subsequent calibration test is not met.
Comparative scheme 4
0.3-0.5% of furfural and 0.04% of ethyl maltol are added into the stationary liquid 1 in the embodiment 1, the rest is kept unchanged, the obtained chicken red blood cell nucleus is stained by 5 mu g/ml of propidium iodide, and the peak becomes 8 by detection of a flow cytometer, the proportion does not meet the requirement, and the single-peak CV values are respectively between 2.2% and 2.5%, so that the requirement of a subsequent calibration test is not met.
Comparative scheme 5
0.3-0.5% of furfural and 0.09% of ethyl maltol are added into the stationary liquid 1 in the embodiment 1, the rest is kept unchanged, the obtained chicken red blood cell nucleus is stained by 5 mu g/ml of propidium iodide, and the peak is formed by detection of a flow cytometer, the proportion is not in accordance with requirements, and the single-peak CV values are respectively between 2.6% and 2.9%, so that the requirements of subsequent calibration tests are met.
In summary, the test results for the 6 sets of embodiments are tabulated as follows:
from the above table, it can be seen that the nucleus with controllable aggregation state and long-term preservation can be obtained from the nucleated red blood cell sample by the nucleus preparation method, and particularly, the obtained nucleus meets the test requirements better after furfural and ethyl maltol are added into the stationary liquid 1.
The results of the tests of the comparative examples of the groups of examples 1 and 5 are tabulated as follows:
it can be seen from the above two tables that in the comparative example 1, when furfural is not added to the fixing solution 1, nuclei meeting the test requirements cannot be prepared, and in the subsequent comparative example, when furfural and ethyl maltol are not added in the concentration range described in the embodiment 6, the prepared nuclei meet the test requirements, but the test effect of the embodiment 6 is far from good, so it is inferred that the test effect of adding 0.3-0.5% of furfural and 0.05-0.08% of ethyl maltol to the fixing solution 1 is the best, and the best meets the test requirements.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (4)
1. A method for producing a cell nucleus with a controllable aggregation state and capable of being preserved for a long period of time, comprising:
s1, collecting a nucleated cell sample, washing, centrifuging to remove a supernatant, and adding a lysis solution for lysis;
s2 adding sucrose solution, centrifuging at 15000-40000g and 4 ℃;
s3, taking the supernatant, adding the obtained cell nucleus into a pre-cooled stationary liquid 1 at the temperature of-20 ℃ for crosslinking;
s4, after crosslinking for 2 days, replacing the stationary liquid 1 with the stationary liquid 2 to obtain the cell nucleus with controllable aggregation state and long-term preservation;
wherein the fixing liquid 1 includes:
methanol or ethanol, which is used for fixing chromatin in cell nucleus, protecting DNA in the chromatin to be stable and realizing aggregation of cell nucleus;
bovine serum albumin or fetal bovine serum or glycerol, which prevents the nuclear membrane from breaking during concussion;
0.05-0.08% of ethyl maltol, which can prolong the storage period;
0.3-0.5% of furfural, and strong sterilizing capability;
water;
the fixing liquid 2 includes:
glutaraldehyde or formaldehyde, wherein glutaraldehyde or formaldehyde is mainly used for maintaining the aggregation state of cell nuclei;
bovine serum albumin or fetal bovine serum or glycerol;
water;
the sucrose solution comprises 1.5-2.2M of sucrose, 0.5-2% of glycerol, 0.1-0.5% of glycine and 0.01M of PBS buffer solution, the concentration and the centrifugation speed of the sucrose solution of 1.5-2.2M ensure the separation of cell nucleus and cytoplasmic membrane components, and protect the integrity of the nuclear membrane and DNA therein in the process; glycerol is a nuclear membrane stabilizer, and glycine has a protective effect on the nuclear membrane stability.
2. The method according to claim 1, wherein the lysate comprises 0.1-0.5% Triton X-100, 0.1-2 μ M dithiothreitol, 0.1-1 μ M phenylmethylsulfonyl fluoride, 0.5-2% ethylphenylpolyethylene glycol, 30-60mM sodium citrate or tris (hydroxymethyl) aminomethane, and 0.01-0.1(v/v)% DMSO.
3. The method according to claim 1, wherein the fixative 1 comprises 70-80% methanol or ethanol, 1-10% bovine serum albumin or fetal bovine serum or glycerol.
4. The method according to claim 1, wherein the fixative 2 comprises glutaraldehyde 2% or formaldehyde 1%, bovine serum albumin 1-10% or fetal bovine serum or glycerol 1%.
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| CN108871897A (en) * | 2018-07-10 | 2018-11-23 | 南京林业大学 | A method of quickly preparing flow cytometry plant sample |
| CN109628555A (en) * | 2019-01-30 | 2019-04-16 | 杭州瑞普基因科技有限公司 | People suitable for unicellular sequencing freezes tumor tissue cell's core separation method |
| CN113444678A (en) * | 2020-03-26 | 2021-09-28 | 武汉华大医学检验所有限公司 | Method for preparing single cell nuclear suspension, single cell sequencing method and kit |
| CN112662737B (en) * | 2020-12-31 | 2021-08-24 | 广州基迪奥生物科技有限公司 | Preparation method and application of animal cell nucleus suspension |
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Application publication date: 20170620 Assignee: Jinan National Science Hygiene Technology Co., Ltd. Assignor: Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences Contract record no.: 2019320010021 Denomination of invention: Method for preparing cell nucleus with controllable aggregation state and capacity of being preserved for a long time License type: Exclusive License Record date: 20190515 |
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