CN103529202A - Method for reducing spontaneous mutation background of wild type human-rat hybridoma AL cells - Google Patents
Method for reducing spontaneous mutation background of wild type human-rat hybridoma AL cells Download PDFInfo
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- CN103529202A CN103529202A CN201310419228.XA CN201310419228A CN103529202A CN 103529202 A CN103529202 A CN 103529202A CN 201310419228 A CN201310419228 A CN 201310419228A CN 103529202 A CN103529202 A CN 103529202A
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Abstract
The invention discloses a method for reducing a spontaneous mutation background of wild type human-rat hybridoma AL cells. The method comprises the following steps: collecting wild type cells; combining with a fluorescent antibody; preparing a sampling suspension solution; sorting the cells; and preserving. The method is simple to operate; the whole process from the collection of the cells and the combination with specific fluorescent proteins to sorting only needs 4-5 hours; continuously-cultured un-mutant (CD59<+>) cells and mutant (CD59<->) cells can be obtained at the same time so that the efficiency of detecting CD59 gene mutation can be improved greatly. With the adoption of the method, the spontaneous mutation rate of the AL cells can be reduced and about 30 mutant cells exist in 105 survival cells and are reduced by two times as much as the spontaneous mutation after Panning, so that the experiment sensitivity of a mutation detection system is improved effectively.
Description
Technical field
The present invention relates to a kind of mammalian cell sudden change detection technique field, in particular a kind of reduction wild type people murine hybridoma A
lthe method of cell spontaneous mutation background.
Background technology
One of key factor of cancer generation is not only in gene mutation, and is the important evidence of environmental contaminants Genotoxic Assessment.A
lcell is Chinese hamster Chinese hamster ovary celI gly
-gained immortalized cells after A mutant strain and the hybridization of normal person fibroblast, No. 11 chromosomes that contain a whole set of Chinese hamster CHO-K1 chromosome and a people.People's No. 11 chromosome codified various kinds of cell surface proteins, comprising the CD59 cell surface antigen (being once called as the antigen into S1) of CD59 gene (being once called as the gene for MIC1) coding.Due to wild type A
lduring cell CD59 gene normal expression, at cell surface, form CD59 antigen, in the situation that rabbit anteserum complement exists, after this antigen is combined with specific antibody E7.1, causes wild-type cell cracking and cannot survive; And mutant cell is because not expressing CD59 albumen, cannot be survived with antibody and role of complement, on double dish, form macroscopic mutant cell clone.According to this feature, A
lcell has been widely used in environmental contaminants as genetoxic detection and Study on Correlative Mechanisms such as radon gas, asbestos, organic contaminant, heavy metals.
Yet, in continuous passage process, wild type A
lthe spontaneous mutation cell of cell can constantly increase, and causes spontaneous mutation background to rise, and the sensitivity of detection in Gene Mutation declines, in order to overcome this phenomenon, and before mutating experiment, wild type A
lcell need adopt Panning technology, removes CD59
-negative cells, to reach the object that reduces sudden change background.This process is specific as follows:
(1) goat anti-mouse IgM processes common plate: 10ml IgM/PBS(20 μ g/ml) IgM is dissolved in PBS) add in the common plate of 100mm, at the bottom of making it be covered in ware uniformly.Room temperature, static 2 hours.PBS cleans after 2 times, adds 5ml1%FBS/PBS (1% calf Trace element is dissolved in PBS), is stored in 4 ℃ (can spend the night) in dark;
(2) A
lcell harvesting: the A in exponential phase
lcell enzymolysis, is suspended in 2%FBS/PBS(2% calf Trace element and is dissolved in PBS) in.Counting, centrifugal, adjust cell density to 2 * 10
6/ ml.Cell is suspended in the 2%FBS/PBS that contains 0.2%CD59 antibody, ice bath 30 minutes.Low-temperature centrifugation, the 2%FBS/PBS washing of precooling 3 times, and by cell dilution to 5 * 10
5/ ml.
(3) cell and goat anti-mouse IgM interact: discard 1%FBS/PBS in plate, the cell seeding that 5ml is suspended in 2%FBS/PBS moves into plate.Hatch 2 hours for 4 ℃.Careful sucking-off supernatant, with careful the washing 3 times of 2%FBS/PBS of precooling, removes and there is no adherent cell and CD59
-spontaneous mutation cell.
(4) mutant cell is not preserved and is detected: add fresh F12 complete culture solution, Continuous Cultivation 3-4 days, collects CD59
+cell, frozen in liquid nitrogen.Meanwhile, need to be to the cell of the collecting background detection that suddenlys change, this process need 14 days.If sudden change detection background is still higher, need to repeat above-mentioned steps.Generally, cell spontaneous mutation background can be down to every 10
5in survivaling cell, there is 70-80 mutant cell.Therefore the method operating process is loaded down with trivial details, one-period needs 3 time-of-weeks conventionally, particularly with IgM, processes plate and clean mutant cell process that the skill level of experiment operator technology be there are certain requirements.Meanwhile, distinct antibodies-goat anti-mouse IgM antibody price of using in experiment is comparatively expensive, and consumption is large.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of reduction wild type people murine hybridoma A is provided
lthe method of cell spontaneous mutation background, based on fluidic cell sorting technology binding specificity fluorescence antibody, screening sudden change and not mutant cell.
The present invention is achieved by the following technical solutions, the present invention includes following steps:
(1) wild-type cell is collected
Collect the A that is cultured to exponential phase
lcell, enzymolysis, digestion, is suspended in F12 complete medium, and after cleaning, making concentration is ± 10
6the single cell suspension of cell/100 μ l;
(2) with the combination of fluorescence antibody
Add after the fluidic cell damping fluid re-suspended cell of 100 μ l precoolings, by matched proportion density, add fluorescently-labeled CD59 specific antibody, 4 ℃ of lucifuge reactions are after 30～60 minutes, centrifugal with the FACS Buffer cleaning of precooling, remove unconjugated antibody;
(3) prepare loading suspension
Re-suspended cell, after the fluidic cell damping fluid of precooling, is sent into flow cell sorter to be measured;
(4) cell sorting
Use flow cell sorter to carry out sorting;
(5) preserve
The not mutant cell amplification that sorting is obtained, liquid nitrogen is preserved.
Described cell comprises A
lcell, the various deficient cell that developed by this cell and the clone of structure.
In described step (1), what enzymolysis, digestion was used is trypsase, for mass content is that 0.25% trypsase and 0.02%EDTA are dissolved in PBS damping fluid and make.
Described step (4) comprises the following steps:
A, set up forward scattering FSC/ lateral scattering SSC scatter diagram, by initial setting door P1, get rid of dead cell and cell fragment, find cell population of interest;
B, set up SSC-W/FSC-W scatter diagram, by secondary, set a door P2, get rid of doublet cell;
C, set up PE-A/FSC-A fluorescence analysis point diagram, according to control cells group set positions yin and yang attribute, define door, define a following negative CD59
-cell mass, defines an above positive CD59
+cell mass.
The cell that described sorting obtains is used for Continuous Cultivation, or as the positive CD59 in CD59 gene flow cytometer detection mutating technology
+with negative CD59
-control cells
The A that the present invention uses
lcell derived is disclosed in Genetics of Somatic Mammalian Cells:Lethal Antigens as Genetic Markers for Study of Human Linkage Groups by THEODORE T.PUCK etc., Proc.Nat.Acad.Sci.USA Vol.68, No.12, pp.3102-3106, December1971.The science of heredity of mammalian somatic cell: fatal antigen is studied mankind's linked gene group as genetic marker.Institute of periodical Proc.Nat.Acad.Sci. full name Wei《 NAS periodical ", be called for short PNAS.This piece of article is published in the 12nd phases 68 volume in November, 1971 PNAS magazine, page number 3102-3106.
The present invention has the following advantages compared to existing technology:
1, the present invention's background of suddenling change is lower: generally by traditional Panning technology background sudden change value, can be reduced to every 10
5in survivaling cell, there is 70-80 sudden change (CD59
-) cell, and by this method, can further reduce sudden change background, reach every 10
5in survivaling cell, approximately there are 30 mutant cells;
2, efficiency is high: general classic method complex steps, and cell is lost more after multistep is processed, and cells up to a million are only several ten thousand cells through antibody screening yield.And adopting airflow classification technology, detection limit is per second reaches as high as 1 * 10
4individual cell, sorting number is per second reaches as high as 500 cells, in one hour, just can sub-elect up to a million CD59
+cell;
3, the cycle is short: the whole process of conventional P anning technology is more loaded down with trivial details, one-period needs 3 time-of-weeks conventionally, particularly with IgM, process plate and clean mutant cell process the skill level of experiment operator technology be there are certain requirements, this method from collecting cell, with specificity fluorescent protein combination, sorting, whole process only needs 4-5 hour, simple and quick and be not subject to the interference of experimental situation condition, and can obtain simultaneously can Continuous Cultivation not sudden change (CD59
+) cell and sudden change (CD59
-) cell, this improves the efficiency of CD59 detection in Gene Mutation to a great extent;
4, experimental expenses is low: the goat anti-mouse IgM antibody using in conventional P anning technology is expensive, antibody consumption is large, and the fluorescence antibody using in this method is comparatively cheap, and each experimental measuring is considerably less, has saved largely experimental expenses.
Accompanying drawing explanation
Fig. 1 is FSC/SSC scatter diagram;
Fig. 2 is SSC-W/FSC-W scatter diagram;
Fig. 3 is PE-A/FSC-A negative cells group fluorescence analysis point diagram;
Fig. 4 is PE-A/FSC-A positive cell group fluorescence analysis point diagram;
Fig. 5 is the airflow classification A that do not suddenly change
lcell parameters arranges figure;
Fig. 6 is that cloning detects airflow classification front and back A
lcell spontaneous mutation background comparison diagram;
Fig. 7 is separate sources A
lcell spontaneous mutation rate comparing result.
Embodiment
Below embodiments of the invention are elaborated, the present embodiment is implemented take technical solution of the present invention under prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
The present embodiment comprises the following steps:
(1) wild-type cell is collected
Collect the A that is cultured to exponential phase
lcell, enzymolysis, digestion, is suspended in F12 complete medium, and after cleaning, making concentration is ± 10
6the single cell suspension of cell/100 μ l;
(2) with the combination of fluorescence antibody
Add after the fluidic cell damping fluid FACS Buffer re-suspended cell of 100 μ l precoolings, by matched proportion density, add fluorescently-labeled CD59 specific antibody, 4 ℃ of lucifuge reactions are after 30～60 minutes, centrifugal with the FACS Buffer cleaning of precooling, remove unconjugated antibody; Fluorescence antibody concentration: a common experiment sample is ± 10
6cell/100 μ l suspension, every 100 μ l cell suspensions add 10-20 μ l fluorescence antibody;
(3) prepare loading suspension
Re-suspended cell is after the FACS of precooling Buffer, FACS Buffer is fluidic cell damping fluid, fills a prescription to be: the bovine serum albumin(BSA) that mass ratio is 1%, and 10mM Sodium azide is dissolved in phosphate buffer PBS(and contains calcium and magnesium ion, pH=7.0), send into flow cell sorter to be measured;
(4) cell sorting
Use flow cell sorter to carry out sorting; The instrument that cell sorting adopts is BD FACS Aria
tMiII Cell Sorter;
A, as shown in Figure 1, while obtaining data on flow cytometer, sets up forward scattering FSC/ lateral scattering SSC scatter diagram, and SSC is used for detecting cell interior granularity, and FSC is used for detecting the size of cell.By initial setting door P1, get rid of dead cell and cell fragment, find cell population of interest, and regulate each photomultiplier device to determine that all cell masses that will analyze are all in the visual range of point diagram.Specific as follows: by arranging and regulate FSC threshold value, the ELIMINATION OF ITS INTERFERENCE that most cell fragment, bubble and laser background are made an uproar (all should be located at FSC-low/SSC-low region) outside analyzed area, next in FSC/SSC point diagram, iris out the peripheral region (P1) of cell population of interest, this region can be called as " P1-FSC/SSC ", it is mainly to draw a circle to approve its scope according to the most concentrated part of cell that P1 establishes door, gets rid of cell granulations or the fragment impurity of excessive too small dispersion around;
B, set up SSC-W/FSC-W scatter diagram, by secondary, set a door P2, get rid of doublet cell, the cell that guarantees finally to carry out fluoroscopic examination and sorting is individual cells, and as shown in Figure 2, W refers to collect the width of signal.SSC-W/FSC-W full name is lateral scattering angle-width/forward-scattering angle-width scatter diagram.Width (as SSC-W) is commonly used to distinguish doublet cell.Because the resulting signal of doublet cell can be larger than individual cells, reaction is ordinate and the large more discrete dotted region of abscissa value on SSC-W/FSC-W figure, and therefore according to the width secondary of the size of cell and granularity, establishing a P2 gets rid of doublet;
C, set up PE-A/FSC-A fluorescence analysis point diagram, as shown in Figure 3 and Figure 4, according to control cells group set positions yin and yang attribute, define door, define a following negative CD59
-cell mass, defines an above positive CD59
+cell mass, defines the door negative (CD59 in following Q4 district in Fig. 3
-) cell mass, in Fig. 4, define the door positive (CD59 in above Q2 district
+) cell mass, in these two groups of cells, must comprise some false positives/false-negative cell or impurity, can define the above or following minute favored area of door by fine adjustment, and regulate a minute lectotype to obtain the highly purified positive and negative control cell, dyestuff-phycoerythrin that PE is antibody coupling; A is the area of fluorescent pulse, and area integral more can accurate response than the height H of fluorescent pulse detects the content of index.PE-A/FSC-A full name is fluorescent dye-fluorescent pulse area/cell size-fluorescent pulse areal analysis point diagram.
It is according to positive and negative control cell group, to account for respectively the number percent division of collecting cell that yin and yang attribute defines door, by arranging and regulate magnitude of voltage and the four-quadrant door position of the FL2 fluorescence channel that PE is corresponding, make the positive that detects and negative cells purity all reach respectively 99.9% and more than, at this moment corresponding quadrant door position is exactly the door that defines of dividing yin and yang attribute control cells.
Fig. 5 represents to obtain by sorting the CD59 of low sudden change background
+cell, door (shown in dotted line) the above Q2 that defines setting according to control cells in figure is partly CD59
+positive cell group, define the following Q4 of door and be partly CD59
-negative cells group.Q2 part all can be used as a minute favored area, defines a He Shemen region, position (shown in arrow and square frame) further improve separating purity by adjusting.In figure, P3 is partly the cell mass of final sorting.
(5) preserve
The not mutant cell amplification that sorting is obtained, liquid nitrogen is preserved.
As shown in Figure 6, for the A after the present embodiment sorting
lcell, detects airflow classification front and back A by cloning
lcell spontaneous mutation background, the left side is the A before sorting
lcell, the right is the A after sorting
lcell, from as can be seen from Figure, airflow classification can significantly reduce background sudden change number.
Cloning detects principle: wild type A
lduring cell CD59 gene normal expression, at cell surface, form CD59 antigen, in the situation that rabbit anteserum complement exists, after this antigen is combined with specific antibody E7.1, causes wild-type cell cracking and cannot survive; And mutant cell is because not expressing CD59 albumen, cannot be survived with antibody and role of complement, on double dish, form macroscopic mutant cell clone.
Detailed process:
1, will process rear cell Continuous Cultivation 7-14 days again, and make the survivaling cell after processing have time enough to recover from the temporary transient dead state of growing, meanwhile, also make the no longer residual CD59 antigen in progeny cell surface of mutant cell breeding:
2, add antibody complement and CD59 AI Continuous Cultivation 7 to 10 days, until form naked eyes, clone as seen, discard nutrient solution, fixing, Giemsa dyeing, clone's number of calculating survivaling cell;
3, statistics mutant cell clone number, calculates cell mutation rate.Clone's number/calibration rear every 1 * 10 of cell mutation rate=mutant cell
5the cell number of survival.
Fig. 7 is to separate sources A
lthe result that cell spontaneous mutation rate contrasts.Wild type A
lcell is 124/10
5cells; By Panning technology, A
lcell spontaneous mutation rate can be down to 80/10
5cells, and airflow classification technology further reduces sudden change background, reaches 30/10
5cells.
Claims (5)
1. one kind is reduced wild type people murine hybridoma A
lthe method of cell spontaneous mutation background, is characterized in that, comprises the following steps:
(1) wild-type cell is collected
Collect the cell that is cultured to exponential phase, enzymolysis, digestion, is suspended in F12 complete medium, and after cleaning, making concentration is ± 10
6the single cell suspension of cell/100 μ l;
(2) with the combination of fluorescence antibody
Add after the fluidic cell damping fluid re-suspended cell of 100 μ l precoolings, by matched proportion density, add fluorescently-labeled CD59 specific antibody, 4 ℃ of lucifuge reactions are after 30～60 minutes, centrifugal with the FACS Buffer cleaning of precooling, remove unconjugated antibody;
(3) prepare loading suspension
Re-suspended cell, after the fluidic cell damping fluid of precooling, is sent into flow cell sorter to be measured;
(4) cell sorting
Use flow cell sorter to carry out sorting;
(5) preserve
The not mutant cell amplification that sorting is obtained, liquid nitrogen is preserved.
2. a kind of reduction wild type people murine hybridoma A according to claim 1
lthe method of cell spontaneous mutation background, is characterized in that, described cell comprises A
lcell, the various deficient cell that developed by this cell and the clone of structure.
3. a kind of reduction wild type people murine hybridoma A according to claim 1
lthe method of cell spontaneous mutation background, is characterized in that, in described step (1), what enzymolysis, digestion was used is trypsase, for mass content is that 0.25% trypsase and 0.02%EDTA are dissolved in PBS damping fluid and make.
4. a kind of reduction wild type people murine hybridoma A according to claim 1
lthe method of cell spontaneous mutation background, is characterized in that, described step (4) comprises the following steps:
A, set up forward scattering FSC/ lateral scattering SSC scatter diagram, by initial setting door P1, get rid of dead cell and cell fragment, find cell population of interest;
B, set up SSC-W/FSC-W scatter diagram, by secondary, set a door P2, get rid of doublet cell;
C, set up PE-A/FSC-A fluorescence analysis point diagram, according to control cells group set positions yin and yang attribute, define door, define a following negative CD59
-cell mass, defines an above positive CD59
+cell mass.
5. a kind of reduction wild type people murine hybridoma A according to claim 1
lthe method of cell spontaneous mutation background, is characterized in that, the cell that described sorting obtains is used for Continuous Cultivation, or as the positive CD59 in CD59 gene flow cytometer detection mutating technology
+with negative CD59
-control cells.
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| CN108486212A (en) * | 2018-04-28 | 2018-09-04 | 中国科学院合肥物质科学研究院 | A kind of method of antagonism heavy metal arsenic bio-toxicity |
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