CN110885778A - Method for separating Pig-a gene mutation cell from in vitro cultured cell - Google Patents

Method for separating Pig-a gene mutation cell from in vitro cultured cell Download PDF

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CN110885778A
CN110885778A CN201811044150.7A CN201811044150A CN110885778A CN 110885778 A CN110885778 A CN 110885778A CN 201811044150 A CN201811044150 A CN 201811044150A CN 110885778 A CN110885778 A CN 110885778A
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cells
pig
magnetic beads
dye
ice
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李若婉
黄鹏程
周长慧
常艳
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Shanghai Yinuosi Biotechnology Ltd By Share Ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract

The invention discloses a method for separating Pig-a gene mutation cells from in vitro cultured cells and application thereof. The method comprises the following steps: (1) mixing an immunofluorescent dye-labeled anti-GPI anchor protein antibody and a magnetic bead against the immunofluorescent dye with the cells; (2) and separating the cells marked by the magnetic beads from the cells not marked by the magnetic beads, wherein the cells marked by the magnetic beads are negative cells of Pig-a gene mutation, and the cells not marked by the magnetic beads are positive cells of Pig-a gene mutation. The method has high separation efficiency for removing Pig-a positive cells prestored in cells, such as human lymphoblastic TK6 cells, can separate 1000 ten thousand cells within 15 minutes, and has simple operation and low cost.

Description

Method for separating Pig-a gene mutation cell from in vitro cultured cell
Technical Field
The invention belongs to the field of biological cells, and particularly relates to a method for separating Pig-a gene mutation cells from in vitro cultured cells.
Background
The Pig-a gene is located in the short arm of the human X chromosome and is involved in the early synthesis of Glycosylphosphatidylinositol (GPI) connexin. In a genotoxicological assay, the Pig-a gene mutation assay can be used to evaluate the ability of a compound to induce a gene mutation. The Pig-a gene mutation test is to detect the expression of GPI anchor protein (such as CD59 and CD55 protein) on the surface of a cell by Flow Cytometry (FCM), and evaluate the capability of a test object to induce gene mutation by the change of the mutation rate of the Pig-a gene.
The existing Pig-a gene mutation method mainly utilizes an animal gene mutation test taking rats as test models. The Pig-a gene mutation method of in vitro cultured animal cells is still under development and establishment. In vitro Pig-a gene mutation test, human lymphoblastic TK6 cells or mouse lymphoma L5178Y cells cultured in vitro are mainly used as test materials, and after treatment of a test object or solvent control and the like, the surface antigen expression of CD55 and CD59 on the cell surface is detected to evaluate whether the Pig-a gene has gene mutation. Cells cultured in vitro accumulate more pre-existing spontaneously mutated cells of the Pig-a gene after prolonged culture. The gene mutation is a low-frequency mutation, and the spontaneous mutation rate of the gene mutation is generally (1-75) multiplied by 10-6. Even after induction by mutagens, the mutation rate is still low. Therefore, in the in vitro Pig-a assay, a cell line with a lower basal mutation value is required to improve the sensitivity of the assay. It is particularly important to eliminate Pig-a gene mutant cells pre-existing in TK6 or L5178Y cells subjected to long-term in vitro subculture.
In the prior art, the method for removing the pre-stored Pig-a mutant cells mainly comprises three methods of sorting Pig-a non-mutant cells by a flow cytometer after immunofluorescence labeling, adsorbing non-Pig-a mutant cells by an antigen coated plate and sorting by a limiting dilution cloning method. The flow sorting technology can realize various sorting tasks, and in the experiment, the flow sorting has the defect of high sorting cost; from 10 with a mutation rate of 30%7The Pig-a mutant cells in the cells need more than 1 hour for separation, and the cost of one-time sorting is about thousands of yuan, so the cost is high. The method of the plate coating antigen adsorption has low sorting efficiency and long screening time, and more mutant cells are likely to remain. The limited dilution cloning method is time-consuming and labor-consuming, the operation of culturing and forming clones is complex, wherein the process of only cloning and culturing needs more than ten days, and the subsequent identification of different clones also needs to be carried outConsuming time and labor.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for separating Pig-a mutant cells from in vitro cultured cells and application thereof, aiming at the defects of high cost and low efficiency of a method for removing prestored Pig-a mutant cells in the prior art. The method can remove cells with higher separation efficiency, such as Pig-a positive cells prestored in human lymphoblastic TK6 cells, and can separate 1000 ten thousand cells within 15 minutes; and the operation is simple and the cost is low.
The immunomagnetic beads are superparamagnetic micronuclei, and can be connected with specific antigen or fluorescein after immune antibody coupling. The present inventors creatively applied this technique to the isolation of Pig-a mutant cells.
The invention mainly solves the technical problems by the following technical means:
the invention provides a method for separating Pig-a gene mutant cells from in vitro cultured cells, which comprises the following steps:
(1) mixing an immunofluorescent dye-labeled anti-GPI anchor protein antibody and a magnetic bead against the immunofluorescent dye with the cells;
(2) and separating the cells marked by the magnetic beads from the cells not marked by the magnetic beads, wherein the cells marked by the magnetic beads are negative cells of Pig-a gene mutation, and the cells not marked by the magnetic beads are positive cells of Pig-a gene mutation.
The immunofluorescent dye described in step (1) of the above method may be PE. The PE is called phycoerythrorin in all, namely: phycoerythrin; is an immunofluorescent dye. It can be replaced by other fluorescent dyes, for example, the labeled antibody is changed into an antibody of FITC-anti-GPI anchor protein, and the magnetic beads only need to use anti-FITC microbeads.
In order to achieve better binding of the immunofluorescent-dye-labeled anti-GPI anchor antibody to GPI anchor protein on cells, in step (1), it is preferable to add magnetic beads against the immunofluorescent dye after mixing the cells with the immunofluorescent-dye-labeled anti-GPI anchor protein antibody. Wherein said mixing is preferably by adding said cells to said anti-GPI-anchor antibody.
After said mixing, and before the addition of the magnetic beads against said immunofluorescent dye, preferably further performing an incubation on ice so that the anti-GPI-anchored-protein antibody can be sufficiently bound to GPI-anchored-protein on the cells; in order to make the magnetic beads against the immunofluorescent dye and the immunofluorescent dye be fully combined and simultaneously avoid the influence of illumination on the immunofluorescent dye, abnormal quenching in the experimental process and cell viability maintenance, after the magnetic beads against the immunofluorescent dye are added, incubation on ice in a dark place is preferably carried out. The time for the incubation on ice or the incubation in the dark on ice is preferably 20-40 min, more preferably 25-35 min, and most preferably 30 min; after intensive research and study, the inventor finds that the ice incubation or the ice dark incubation within the time period can ensure that the anti-GPI-anchor-protein antibody and the GPI-anchor-protein on the cell and the magnetic bead of the anti-immunofluorescent dye and the immunofluorescent dye can be fully combined, can avoid the influence on the cell activity caused by too long incubation time, and finally ensures the accuracy and the effectiveness of the method.
Preferably, after the incubation on ice or the incubation on ice in the dark place, the cells are washed by PBS, the washing method is centrifugation, the speed of the centrifugation is preferably 800-1500 rpm, more preferably 1000rpm, and the time of the centrifugation is preferably 3-8 min, more preferably 5 min; the PBS preferably contains 2% bovine serum albumin.
The relative amount of the cells in step (1) of the above method and the immunofluorescent dye-labeled anti-GPI anchor protein antibody is preferably 24 to 40. mu.L/2X 106And (4) cells.
The GPI-anchor protein described in step (1) of the above method is preferably CD55 and/or CD 59.
The cells described in step (1) of the above method may be human lymphocytes; among them, human lymphoblastic TK6 cell is widely applicable as described above, so the human lymphocyte mentioned in the present invention is exemplified by human lymphoblastic TK6 cell.
In step (2) of the above method, the separation is preferably performed by using a magnetic bead sorting technique; preferably, the separation is performed using a magnetic column and a magnet holder.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the method has higher separation efficiency for removing the Pig-a positive cells prestored in the cells (such as TK6 cells), and can separate 1000 ten thousand cells within 15 minutes; and the operation is simple and the cost is low.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
1. Abbreviations
BSA: bovine albumin
PS: antibody staining solution buffer solution
PBS: phosphate buffered saline solution
2. Materials and reagents:
PE-Mouse-Anti-Human CD55 antibody (BD Biosciences, cat # 341030), PE-Mouse-Anti-Human CD59 antibody (BD Biosciences, cat # 560953), Anti-PE magnetic beads (Miltenyi Biotec, cat # 130-TMSeparator). Sterile PBS (gibco, cat # 10010-023), horse serum (gibco, 26050088), paraformaldehyde (sigma, cat # 158127-5G), 0.22 μm microfiltration membrane (Millipop, cat # SLGP033RB), bovine albumin (national drug group chemical Co., Ltd., cat # 69003436).
3. Preparation of reagents
Dye liquor buffer (PS): 120mg BSA was dissolved in 6mL PBS.
Antibody staining solution: prepared by mixing 72 mu LCD55-PE, 120 mu LCD59PE and 345 mu L of dye solution buffer (PS) prepared by the method.
Fixing liquid: 3mL of PBS and 3mL of 4% formaldehyde solution are mixed to prepare a 2% formaldehyde solution, wherein the mass fraction is the percentage.
After the preparation of the reagent is finished, filtering the mixture by using a 0.22 mu m microporous filter membrane, and storing the filtered mixture on ice for later use.
EXAMPLE 1 isolation of Pig-a Gene-mutated cells
1. Counting cells in logarithmic phase, taking 600 ten thousand cells, dividing into three parts, centrifuging at 1000RPM for 5min, discarding supernatant, adding 10mL of PBS precooled at 2-8 ℃, and washing once. The supernatant was centrifuged again and approximately 10. mu.L of liquid resuspended cells remained in each tube.
2. 175 μ L of antibody stain was added to a 1.5mL conical centrifuge tube, the cell suspension was gently added to the antibody stain and gently mixed to allow all cells to contact the antibody, preventing the cells from unmarked by the antibody and causing false positives. Incubate for 30 + -5 min on ice in the dark. 2mL of pre-cooled PBS containing 2% BSA was added, the supernatant was discarded after centrifugation, and approximately 10. mu.L of liquid was left per tube to resuspend the cells.
3. Adding 150 mu L of anti-PE-magnetic beads into a 1.5mL pointed-bottom centrifuge tube, gently adding the cell suspension into the dye solution, gently mixing uniformly, then carrying out incubation on ice for 30 +/-5 min in a dark place. Each tube was washed 1 time with 2mL of pre-cooled PBS containing 2% BSA. And adding 500 mu L of PBS into one part of the mixture, re-suspending, adding 500 mu L of PBS solution containing 2% formaldehyde solution, incubating on ice for 5-10 min, and detecting the proportion of Pig-a gene mutation positive cells by using a flow cytometer. The value is the mutation rate of the basic Pig-a gene positive cells before removal.
4. And centrifuging the other two cells marked with anti-PE-magnetic beads, discarding supernatant, re-suspending the cells by using 1mL of precooled PBS, and separating the positive and negative cells marked by the magnetic beads by using a Meitian whirling magnetic column and a magnet rack in a sterile environment. The magnetic column was clamped in a magnet and 5mL of cold PBS was added to rinse the magnetic column. 1mL of the cell suspension was added and the filtrate was discarded. The magnetic column was then removed from the magnet, 5mL of pre-cooled PBS was added, and the eluted magnetic column-adsorbed cells were collected.
5. Centrifuging to remove supernatant, adding 1mL of precooled PBS into each tube for resuspension, taking 500 mu L of cell suspension, adding 500 mu L of PBS solution containing 2% formaldehyde solution into one tube, incubating on ice for 5-10 min, and detecting the mutation rate of the Pig-a gene mutation positive cells by using a flow cytometer.
6. Repeating steps 5 and 6, using another tube of cells without fixation, isolating again and detecting the basal Pig-a gene mutation rate.
7. Collecting the mutation rate of the basic Pig-a gene at (1-75) x 10-6The cells inside are centrifuged, resuspended by using a complete culture medium, subjected to amplification culture and frozen into liquid nitrogen for later use.
The results are shown in Table 1:
TABLE 1 results of experiments on removal of Pig-a positive cells pre-stored in TK6 cells by magnetic column separation
Pig-a Gene mutation-Positive cell ratio (10)-6)
Before separation 237418.0
After one separation 1770.2
After twice separation 39.9
From the above results, it can be seen that: the magnetic bead separation method has high separation efficiency for removing Pig-a positive cells prestored in TK6 cells, 1000 ten thousand cells can be separated within 15 minutes, the operation is simple, the cost of a single separation column is less than 20 yuan, and the separation cost is low.

Claims (10)

1. A method for isolating a Pig-a gene mutant cell from a cell cultured in vitro, comprising the steps of:
(1) mixing an immunofluorescent dye-labeled anti-GPI anchor protein antibody and a magnetic bead against the immunofluorescent dye with the cells;
(2) and separating the cells marked by the magnetic beads from the cells not marked by the magnetic beads, wherein the cells marked by the magnetic beads are negative cells of Pig-a gene mutation, and the cells not marked by the magnetic beads are positive cells of Pig-a gene mutation.
2. The method of claim 1, wherein in step (1), said cells are mixed with said immunofluorescent-dye-labeled anti-GPI anchor protein antibody, followed by addition of magnetic beads against said immunofluorescent dye;
and/or, the separation times in the step (2) are more than 1 time.
3. The method of claim 2, wherein said cells are further incubated on ice after mixing with said immunofluorescent dye-labeled anti-GPI-anchor protein antibody; said mixing is preferably by adding said cells to said anti-GPI-anchor protein antibody;
and/or, after the magnetic beads resisting the immunofluorescent dye are added, incubating on ice in a dark place.
4. The method according to claim 3, wherein the incubation on ice or the incubation on ice in the absence of light is preferably carried out for a period of 20 to 40min, more preferably 25 to 35min, most preferably 30 min; and/or, the ice on the incubation or the ice in the dark after the cell washing with PBS, the PBS preferably containing 2% bovine serum albumin.
5. The method according to claim 4, wherein the washing method is centrifugation, the speed of the centrifugation is preferably 800-1500 rpm, more preferably 1000rpm, and the time of the centrifugation is preferably 3-8 min, more preferably 5 min.
6. The method of claim 1, wherein the relative amount of said cell to said immunofluorescent dye-labeled anti-GPI anchor protein antibody is 24-40 μ L/2 x 106And (4) cells.
7. The method of claim 1, wherein the GPI-anchored protein is CD55 and/or CD 59.
8. A method as claimed in any one of claims 1 to 7 wherein the cells are human lymphocytes such as human lymphoblastic TK6 cells.
9. The method of claim 1, wherein in step (2), the separation is by magnetic bead sorting.
10. The method of claim 9, wherein the separating is performed using a magnetic column and a magnet holder.
CN201811044150.7A 2018-09-07 2018-09-07 Method for separating Pig-a gene mutation cell from in vitro cultured cell Pending CN110885778A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111829940A (en) * 2020-07-23 2020-10-27 四川大学 Flow cytometry detection method for Pig-a gene mutation test in rat body

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JP2013015517A (en) * 2011-06-10 2013-01-24 Teijin Pharma Ltd Method of detecting gpi anchor protein deficient cells
CN105017423A (en) * 2005-08-11 2015-11-04 阿皮·马托西安-罗杰斯 TCR-V-beta peptides for treatment and diagnosis of autoimmune diseases
CN108118029A (en) * 2016-11-30 2018-06-05 深圳宾德生物技术有限公司 The screening technique of high anti-cancer activity T cell and application

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
CN105017423A (en) * 2005-08-11 2015-11-04 阿皮·马托西安-罗杰斯 TCR-V-beta peptides for treatment and diagnosis of autoimmune diseases
JP2013015517A (en) * 2011-06-10 2013-01-24 Teijin Pharma Ltd Method of detecting gpi anchor protein deficient cells
CN108118029A (en) * 2016-11-30 2018-06-05 深圳宾德生物技术有限公司 The screening technique of high anti-cancer activity T cell and application

Non-Patent Citations (2)

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Title
STEPHEN D. DERTINGER等: ""When pigs fly: Immunomagnetic separation facilitates rapid determination of Pig-a mutant frequency by flow cytometric analysis"" *
秦美蓉;郑丹丹;黄泽愉;王晓炜;李俊鹏;王平;: "免疫磁珠分离技术联合流式细胞术检测ENU致大鼠体内Pig-a基因突变" *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111829940A (en) * 2020-07-23 2020-10-27 四川大学 Flow cytometry detection method for Pig-a gene mutation test in rat body
CN111829940B (en) * 2020-07-23 2022-03-22 四川大学 Flow cytometry detection method for rat peripheral blood Pig-a gene mutation test

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