CN103529202B - A method for reducing the spontaneous mutation background of wild-type human-mouse hybridoma AL cells - Google Patents

Abstract

The invention discloses a method for reducing the spontaneous mutation background of wild-type human-mouse hybridoma _AL cells, which comprises the steps of collecting wild-type cells; combining with fluorescent antibodies; preparing sample suspension; cell sorting; and preserving. The method is simple to operate, and the whole process from collecting cells, combining with specific fluorescent proteins to sorting, only takes 4-5 hours, and can simultaneously obtain unmutated (CD59 ⁺ ) cells and mutant (CD59 ^- ) cells that can be continuously cultured , which greatly improves the efficiency of CD59 gene mutation detection. Through this method, the spontaneous mutation rate of _AL cells can be reduced to about 30 mutant cells in 10 ⁵ surviving cells, which is 2 times lower than the spontaneous mutation obtained after Panning, which effectively improves the experimental sensitivity of the mutation detection system.

Description

A method for reducing the spontaneous mutation background of wild-type human-mouse hybridoma AL cells

technical field

The invention relates to the technical field of mammalian cell mutation detection, in particular to a method for reducing the spontaneous mutation background of wild-type human-mouse hybridoma _AL cells.

Background technique

Gene mutation is not only one of the key factors of cancer, but also an important basis for evaluating the genotoxicity of environmental pollutants. _AL cells are immortalized cells obtained by crossing the gly ^- A mutant of Chinese hamster CHO cells with normal human fibroblasts, and contain a complete set of Chinese hamster CHO-K1 chromosomes and a human chromosome 11. Human chromosome 11 can encode a variety of cell surface proteins, including CD59 cell surface antigen (formerly known as S1 antigen) encoded by CD59 gene (formerly known as MIC1 gene). Due to the normal expression of the CD59 gene of wild-type A _L cells, the CD59 antigen is formed on the cell surface. In the presence of rabbit serum complement, the antigen binds to the specific antibody E7.1, causing the wild-type cells to lyse and fail to survive; while the mutation The cells survived because they could not express CD59 protein and could not interact with antibodies and complements, and formed mutant cell clones visible to the naked eye on the culture dish. According to this feature, _AL cells have been widely used in the detection of genotoxicity of environmental pollutants such as radon, asbestos, organic pollutants, heavy metals, and related mechanism research.

However, in the process of continuous passage, the spontaneous mutation cells of wild-type _AL cells will continue to increase, resulting in an increase in the background of spontaneous mutations and a decrease in the sensitivity of gene mutation detection. In order to overcome this phenomenon, before the mutation experiment, wild-type _AL cells Panning technology is required to remove CD59 ^- negative cells in order to reduce the mutation background. The process is as follows:

(1) Goat anti-mouse IgM treatment of ordinary plates: 10ml IgM/PBS (20μg/ml) IgM dissolved in PBS) added to 100mm ordinary plates, so that it evenly covers the bottom of the dish. Room temperature, let stand for 2 hours. After washing twice with PBS, add 5ml of 1% FBS/PBS (1% calf placenta serum dissolved in PBS), and store in the dark at 4°C (overnight);

(2) _AL cell collection: _AL cells in logarithmic growth phase were hydrolyzed, suspended in 2% FBS/PBS (2% calf placental serum dissolved in PBS). Count, centrifuge, and adjust the cell density to 2×10 ⁶ /ml. Cells were suspended in 2% FBS/PBS containing 0.2% CD59 antibody and kept on ice for 30 minutes. Centrifuge at low temperature, wash 3 times with pre-cooled 2% FBS/PBS, and dilute the cells to 5×10 ⁵ /ml.

(3) Interaction between cells and goat anti-mouse IgM: Discard 1% FBS/PBS in the plate, and plant 5ml of cells suspended in 2% FBS/PBS into the plate. Incubate at 4°C for 2 hours. Carefully aspirate the supernatant and wash carefully 3 times with pre-cooled 2% FBS/PBS to remove non-adherent cells and CD59 ^- spontaneous mutant cells.

(4) Preservation and detection of non-mutated cells: add fresh F12 complete culture medium, culture continuously for 3-4 days, collect CD59 ⁺ cells, and freeze them in liquid nitrogen. At the same time, the collected cells need to be tested for mutation background, which takes 14 days. If the mutation detection background is still high, the above steps need to be repeated. In general, the spontaneous mutation background of cells can be reduced to 70-80 mutant cells per 10 ⁵ surviving cells. It can be seen from the above that the operation process of this method is cumbersome, and one cycle usually takes 3 weeks, especially the process of treating the plate with IgM and washing the mutant cells requires a certain degree of proficiency of the experimental operator. At the same time, the special antibody used in the experiment—goat anti-mouse IgM antibody is relatively expensive and used in large quantities.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for reducing the background of spontaneous mutations in wild-type human-mouse hybridoma _AL cells, based on flow cytometry technology combined with specific fluorescent antibodies to screen for mutations and non-mutations cell.

The present invention is achieved through the following technical solutions, and the present invention comprises the following steps:

(1) Collection of wild-type cells

Collect the _AL cells cultured to the logarithmic growth phase, enzymatically digest, suspend in F12 complete medium, wash and make a single cell suspension with a concentration of ±10 ⁶ cells/100 μl;

(2) Binding to fluorescent antibodies

Add 100 μl of pre-cooled flow cytometry buffer to resuspend the cells, add fluorescently labeled CD59-specific antibody according to the proportioned concentration, and react in the dark at 4°C for 30-60 minutes, then wash and centrifuge with pre-cooled FACS Buffer to remove unbound cells. Antibodies;

(3) Preparation of sample suspension

After resuspending the cells in the pre-cooled flow cytometry buffer, send them to the flow cytometry sorter for testing;

(4) Cell sorting

sorting using a flow cytometer;

(5) save

The unmutated cells obtained by sorting were expanded and stored in liquid nitrogen.

The cells include _AL cells, various defective cells developed from the cells, and constructed cell lines.

In the step (1), trypsin is used for enzymatic digestion, which is prepared by dissolving 0.25% trypsin and 0.02% EDTA in PBS buffer.

The step (4) includes the following steps:

a. Establish a forward scatter FSC/side scatter SSC scatter diagram, and preliminarily set the gate P1 to exclude dead cells and cell debris, and find the target cell population;

b. Establish SSC-W/FSC-W scatter diagram, and exclude doublet cells by setting gate P2 twice;

c. Establish the PE-A/FSC-A fluorescence analysis point diagram, set the positive and negative gates according to the position of the control cell population, define negative CD59 ^- cell populations below the gate, and positive CD59 ⁺ cell populations above the gate.

The cells obtained by the sorting are used for continuous culture, or as positive CD59 ⁺ and negative CD59 ^- control cells in CD59 gene flow cytometry detection mutation technology

The _AL cell source used in the present invention 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, December 1971. Genetics of mammalian somatic cells: lethal antigens as genetic markers to study human linked gene groups. The full name of the journal Proc.Nat.Acad.Sci. is "Proceedings of the National Academy of Sciences of the United States", or PNAS for short. This article was published in the November 1971 issue of PNAS Magazine, Volume 12, Volume 68, Pages 3102-3106.

Compared with the prior art, the present invention has the following advantages:

1. The mutation background of the present invention is lower: Generally, the background mutation value can be reduced to 70-80 mutant (CD59 ^- ) cells per 10 ⁵ surviving cells through traditional Panning technology, and through this method, the mutation background can be further reduced , reaching about 30 mutant cells per 10 ⁵ surviving cells;

2. High efficiency: Generally, the steps of traditional methods are cumbersome, and many cells are lost after multi-step treatment, and the yield of millions of cells after antibody screening is only tens of thousands of cells. With flow sorting technology, the detection amount can reach up to 1×10 ⁴ cells per second, and the sorting number can reach up to 500 cells per second, and millions of CD59 ⁺ cells can be sorted within one hour;

3. Short cycle: The whole process of traditional Panning technology is cumbersome, and a cycle usually takes 3 weeks, especially the process of treating the plate with IgM and cleaning the mutant cells has certain requirements for the proficiency of the experimental operator. This method starts from collecting cells, Combined with specific fluorescent protein and sorted, the whole process only takes 4-5 hours, simple, fast and not easily disturbed by experimental environmental conditions, and can simultaneously obtain non-mutated (CD59 ⁺ ) cells and mutant (CD59 ^- ) cells, which greatly improves the efficiency of CD59 gene mutation detection;

4. Low experimental cost: The goat anti-mouse IgM antibody used in the traditional Panning technique is expensive, and the amount of antibody used is large, while the fluorescent antibody used in this method is relatively cheap, and the amount of each experiment is very small, to a great extent Save experiment cost.

Description of drawings

Figure 1 is the FSC/SSC scatter diagram;

Figure 2 is a scatter diagram of SSC-W/FSC-W;

Fig. 3 is the fluorescence analysis point diagram of PE-A/FSC-A negative cell group;

Fig. 4 is the fluorescence analysis point diagram of PE-A/FSC-A positive cell group;

Fig. 5 is a flow cytometric sorting diagram of parameter settings for unmutated _AL cells;

Figure 6 is a background comparison of _AL cell spontaneous mutation before and after flow cytometry detection by cloning method;

Figure 7 is a comparison of the spontaneous mutation rates of _AL cells from different sources.

Detailed ways

The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following implementation example.

This embodiment includes the following steps:

(1) Collection of wild-type cells

Collect the _AL cells cultured to the logarithmic growth phase, enzymatically digest, suspend in F12 complete medium, wash and make a single cell suspension with a concentration of ±10 ⁶ cells/100 μl;

(2) Binding to fluorescent antibodies

Add 100 μl pre-cooled flow cytometry buffer FACS Buffer to resuspend the cells, add fluorescently labeled CD59-specific antibody according to the proportioned concentration, react in the dark at 4°C for 30-60 minutes, wash and centrifuge with pre-cooled FACS Buffer, remove Unbound antibody; fluorescent antibody concentration: usually an experimental sample is ±10 ⁶ cells/100μl suspension, add 10-20μl fluorescent antibody per 100μl cell suspension;

(3) Preparation of sample suspension

After resuspending cells in pre-cooled FACS Buffer, FACS Buffer is a flow cytometry buffer, the formula is: bovine serum albumin with a mass ratio of 1%, 10mM sodium azide dissolved in phosphate buffered saline PBS (containing calcium and magnesium ions, pH=7.0), sent to the flow cytometer for testing;

(4) Cell sorting

Sorting was performed using a flow cytometer; the instrument used for cell sorting was BD FACS Aria ^TM III Cell Sorter;

a. As shown in Figure 1, when acquiring data on the flow cytometer, establish a forward scatter FSC/side scatter SSC scatter diagram, SSC is used to detect the particle size inside the cell, and FSC is used to detect the size of the cell. By initially setting the gate P1, excluding dead cells and cell debris, finding the target cell population, and adjusting each photomultiplier device to ensure that all the cell populations to be analyzed are within the visible range of the dot plot. The details are as follows: By setting and adjusting the FSC threshold, most of the cell debris, air bubbles and laser background noise are excluded from the analysis area (all should be set in the FSC-low/SSC-low area), and then in the FSC/ The surrounding area (P1) of the target cell group is circled in the SSC dot diagram. This area can be called "P1-FSC/SSC". Small dispersed cell particles or debris impurities;

b. Establish the SSC-W/FSC-W scatter diagram, and exclude the doublet cells by setting the gate P2 twice to ensure that the final fluorescent detection and sorting cells are single cells, as shown in Figure 2, W means The width of the collected signal. SSC-W/FSC-W stands for Side Scatter Angle-Width/Forward Scatter Angle-Width Scatter Plot. Width (such as SSC-W) is often used to distinguish doublet cells. Since the signal obtained by the doublet cell will be larger than that of a single cell, the reaction on the SSC-W/FSC-W diagram is a point-like area with large vertical and horizontal coordinates, so according to the size of the cell and the size of the particle The width of the degree is set gate P2 twice to exclude doublets;

c. Establish the PE-A/FSC-A fluorescence analysis point map, as shown in Figure 3 and Figure 4, set the negative and positive limit gates according to the position of the control cell population, the negative CD59 ^- cell population is defined below the gate, and the negative CD59- cell population is defined above the gate. Positive CD59 ⁺ cell population, the Q4 area below the gate is defined as negative (CD59 ^- ) cell population in Figure 3, and the Q2 area above the gate is defined as positive (CD59 ⁺ ) cell population in Figure 4, these two populations of cells must contain some false positives /False-negative cells or impurities can be obtained by precisely adjusting the sorting area defined above or below the gate, and adjusting the sorting mode to obtain high-purity positive and negative control cells. PE is an antibody-coupled dye-phycoerythrin; A is the area of the fluorescence pulse, and the area integral can more accurately reflect the content of the detection index than the height H of the fluorescence pulse. The full name of PE-A/FSC-A is fluorescent dye-fluorescence pulse area/cell size-fluorescence pulse area analysis dot plot.

The positive and negative control gates are divided according to the percentages of the positive and negative control cell populations in the collected cells. By setting and adjusting the voltage value of the FL2 fluorescence channel corresponding to PE and the position of the four-quadrant gate, the purity of the detected positive and negative cells is equal. Reaching 99.9% and above respectively, at this time the corresponding quadrant gate position is the defining gate for dividing negative and positive control cells.

Figure 5 shows the CD59 ⁺ cells with low mutation background obtained by sorting. In the figure, the Q2 part above the defined gate (shown by the dotted line) set by the control cells is the CD59 ⁺ positive cell population, and the Q4 part below the defined gate is the ^CD59- positive cell population. Negative cell population. The Q2 part can be used as a sorting area, and the sorting purity can be further improved by adjusting the position of the defined gate and the area of the gate (shown by the arrow and the box). Part P3 in the figure is the final sorted cell population.

(5) save

The unmutated cells obtained by sorting were expanded and stored in liquid nitrogen.

As shown in Figure 6, for the _AL cells after sorting in this embodiment, the spontaneous mutation background of AL cells before and after flow cytometry sorting was detected by cloning method, the left side is the _AL cells before sorting, and the right side is the sorted _AL cells _AL cells, as can be seen from the figure, flow sorting can significantly reduce the number of background mutations.

The detection principle of the cloning method is: when the CD59 gene of wild-type A _L cells is normally expressed, the CD59 antigen is formed on the cell surface, and in the presence of rabbit serum complement, the antigen binds to the specific antibody E7.1, resulting in the lysis of the wild-type cells Can not survive; mutant cells cannot survive because they cannot express CD59 protein and cannot interact with antibodies and complements, forming mutant cell clones visible to the naked eye on the culture dish.

Specific process:

1. Re-cultivate the treated cells continuously for 7-14 days, so that the surviving cells after treatment have enough time to recover from the temporary stagnation of growth, and at the same time, the CD59 antigen will no longer remain on the surface of the progeny of the mutant cells:

2. Add antibody complement to interact with CD59 antigen and continue to culture for 7 to 10 days until visible clones are formed, discard the culture medium, fix, Giemsa staining, and calculate the number of clones of surviving cells;

3. Count the number of mutant cell clones and calculate the cell mutation rate. Cell mutation rate = clone number of mutant cells/number of surviving cells per 1×10 ⁵ after calibration.

Figure 7 is the result of comparing the spontaneous mutation rate of _AL cells from different sources. The wild-type _AL cells are 124/10 ⁵ cells; through Panning technology, the spontaneous mutation rate of _AL cells can be reduced to 80/10 ⁵ cells, and the flow sorting technology further reduces the mutation background to 30/10 ⁵ cells.

Claims (4)

1. A method for reducing the spontaneous mutation background of wild-type human mouse hybridoma _AL cells, characterized in that, comprising the following steps: (1) Collection of wild-type cells Cells cultured to logarithmic growth phase were harvested, enzymatically digested, suspended in F12 complete medium, washed and made into a single cell suspension with a concentration of ±10 ⁶ cells/100 μl; (2) Binding to fluorescent antibodies Add 100 μl of pre-cooled flow cytometry buffer to resuspend the cells, add fluorescently labeled CD59-specific antibody according to the proportioned concentration, and react in the dark at 4°C for 30-60 minutes, then wash and centrifuge with pre-cooled FACS Buffer to remove unbound cells. Antibodies; (3) Preparation of sample suspension After resuspending the cells in the pre-cooled flow cytometry buffer, send them to the flow cytometry sorter for testing; (4) Cell sorting sorting using a flow cytometer; (5) save The unmutated cells obtained by sorting are expanded and stored in liquid nitrogen; the cells include _AL cells, various defective cells developed from the cells, and cell lines constructed. 2. a kind of method that reduces wild-type human mouse hybridoma _AL cell spontaneous mutation background according to claim 1, is characterized in that, in described step (1), the enzyme used for enzymatic digestion is trypsin, enzyme The buffer used for digestion is PBS buffer with a mass content of 0.25% trypsin and 0.02% EDTA. 3. a kind of method that reduces wild-type human mouse hybridoma _AL cell spontaneous mutation background according to claim 1, is characterized in that, described step (4) comprises the following steps: a. Establish a forward scatter FSC/side scatter SSC scatter diagram, and preliminarily set the gate P1 to exclude dead cells and cell debris, and find the target cell population; b. Establish SSC-W/FSC-W scatter diagram, and exclude doublet cells by setting gate P2 twice; c. Establish the PE-A/FSC-A fluorescence analysis point diagram, set the positive and negative gates according to the position of the control cell population, define negative CD59 ^- cell populations below the gate, and positive CD59 ⁺ cell populations above the gate. 4. A method for reducing the background of spontaneous mutations in wild-type human mouse hybridoma _AL cells according to claim 1, wherein the cells obtained by the sorting are used for continuous culture, or as CD59 gene flow detection Positive CD59 ⁺ and negative ^CD59- control cells in the mutagenesis technique.