CN111004819A

CN111004819A - RAW264.7 single cell stable cell line transfected with red fluorescent protein and screening method thereof

Info

Publication number: CN111004819A
Application number: CN201911417849.8A
Authority: CN
Inventors: 王少雄; 吕品
Original assignee: Shanghai Mab Venture Biological Pharmaceutical Co ltd
Current assignee: Shanghai Mab Venture Biological Pharmaceutical Co ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2020-04-14

Abstract

The invention relates to a RAW264.7 single cell stable cell line for transfecting red fluorescent protein and a screening method thereof. The screening method comprises the following steps: (1) adding pDSRED2-C1 plasmid and transfection reagent into RAW264.7 cells for transfection; (2) applying a screening pressure to the cells obtained in step (1); (3) and (3) selecting the cells in the transfection positive step (2) according to the fluorescence observation result, digesting, resuspending, diluting and plating single cells, and screening the stable single cell strains. The method for screening the RAW264.7 single cell stable cell line transfected with the red fluorescent protein can obtain the RAW264.7 single cell line with good transfection effect, and has good passage stability.

Description

RAW264.7 single cell stable cell line transfected with red fluorescent protein and screening method thereof

Technical Field

The invention belongs to the technical field of bioengineering, particularly relates to a RAW264.7 single cell stable cell line and a screening method thereof, and particularly relates to a RAW264.7 single cell stable cell line transfected with red fluorescent protein and a screening method thereof.

Background

Compared with the green fluorescent protein, the red fluorescent protein has obvious advantages, and firstly, the red fluorescent protein can be used together with GFP to solve some scientific problems which cannot be solved by GFP alone; secondly, the red fluorescent protein has longer excitation and emission wavelength and can cover the wavelength range which cannot be related to GFP; above all, the low background of the red fluorescent protein when the red fluorescent protein is imaged in cells is more suitable for the research of biological science. Because of these advantages of the red fluorescent protein, it is rapidly gaining a large number of applications in biological research.

The stable cell line is constructed by integrating exogenous plasmid DNA into host cell chromosome to make host cell express target protein for a long time. High quality stable cell lines play a very important role in biological research, including gene function research, recombinant antibodies, drug development, and the like.

In the prior art, reports on how to construct and screen a RAW264.7 single cell stable cell line transfected with red fluorescent protein are few, so that the development of a RAW264.7 single cell stable cell line transfected with red fluorescent protein with good transfection effect and good passage stability has important significance for biological research.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a RAW264.7 single cell stable cell line and a screening method thereof, in particular to a RAW264.7 single cell stable cell line transfected with red fluorescent protein and a screening method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for screening RAW264.7 single cell stable cell line transfected with red fluorescent protein, the method comprising:

(1) adding pDSRED2-C1 plasmid and transfection reagent into RAW264.7 cells for transfection;

(2) applying a screening pressure to the cells obtained in step (1);

(3) and (3) selecting the cells in the transfection positive step (2) according to the fluorescence observation result, digesting, resuspending, diluting and plating single cells, and screening the stable single cell strains.

The method for screening the RAW264.7 single cell stable cell line transfected with the red fluorescent protein can obtain the RAW264.7 single cell line with good transfection effect, and has good passage stability.

Preferably, the operation method of step (1) comprises: resuspending RAW264.7 cells in complete medium and plating in cell culture plate for culture; the complete medium was replaced with a serum-free basal medium, to which the pDsRED2-C1 plasmid and transfection reagents were added for transfection.

The pDSRED2-C1 plasmid is a codon-optimized DsRed2 red fluorescent protein plasmid, and the correct sequence of the red fluorescent protein DsRed is confirmed by sequencing.

The transfection reagents of the present invention include

LTX Reagent or

HDTransfect Reagent, the latter transfection was more effective.

The complete culture medium involved in the invention is a DMEM medium containing 10% FBS,

preferably, the plating density of the RAW264.7 cells is (1-10). times.10⁴One/hole, e.g. 1X 10⁴One/hole, 5X 10⁴One/hole or 10X 10⁴Individual/hole, etc., any specific point value within the range can be selected, and is not described in detail herein. Preferably 5X 10⁴Per well.

Preferably, the culturing time is 20-30h, such as 20h, 22h, 24h, 25h, 26h, 28h or 30h, and any specific point value in the range can be selected, and is not repeated herein.

Preferably, the serum-free basal medium is serum-free DMEM medium. Serum-free DMEM media after incubation at 37 ℃ is preferred.

Preferably, the pDSRED2-C1 plasmid is dissolved in serum-free DMEM medium, and the concentration of the pDSRED2-C1 plasmid is 8-12mg/L, such as 8mg/L, 9mg/L, 10mg/L, 11mg/L or 12mg/L, and any specific point value in the range can be selected, and is not repeated herein.

Preferably, the transfection reagent is dissolved in serum-free DMEM medium, and the volume percentage content thereof is 1-3%, for example, 1%, 2%, or 3%, and any specific point value in the range can be selected, which is not described herein again.

Preferably, said applying of a selection pressure to the cells means culturing the cells with a G418 selection medium.

The G418 reagent related to the invention is an aminoglycoside antibiotic, and is the most commonly used resistance screening reagent for stable transfection in molecular genetic tests. It inhibits the gene of transposon Tn601, Tn5, interferes the function of ribosome to block protein synthesis, and produces toxin to prokaryotic and eukaryotic cells, so as to obtain high-efficiency expression of resistance product aminoglycoside phosphotransferase, and make the cells obtain resistance and grow in selective culture medium containing G418. The selection characteristic of G418 has been widely applied in gene transfer, gene knockout, resistance screening, transgenic animals and the like.

Preferably, the concentration of G418 in the G418 screening medium is 800-1000. mu.g/mL, such as 800. mu.g/mL, 850. mu.g/mL, 900. mu.g/mL, 950. mu.g/mL or 1000. mu.g/mL, etc., any specific point value within the range can be selected, and is not repeated herein.

Preferably, the application of the selection pressure to the cells is performed after transfection for 20-25h (e.g., 20h, 21h, 22h, 23h, 24h, 25h, etc.). Wherein, the complete culture medium can be replaced for culture after 4 hours of transfection.

Preferably, the fluorescence observation is performed after applying the screening pressure for 10-15 days (10 days, 11 days, 12 days, 13 days, 14 days, 15 days, etc.).

Preferably, the resuspension refers to resuspending the cells in 400-500. mu.g/mL (400. mu.g/mL, 420. mu.g/mL, 450. mu.g/mL, 480. mu.g/mL, or 500. mu.g/mL, etc.) of G418 selection medium.

Preferably, the single cell dilution plating refers to plating the diluted cells in a 96-well plate.

Preferably, the method for screening the single cell stable cell strain comprises the following steps: and screening single cell wells, culturing and replacing the cells with a G418 screening culture medium, expanding the cells with strong fluorescence signals after 6-10 days (6 days, 7 days, 8 days, 9 days, 10 days and the like), culturing and replacing the cells with the G418 screening culture medium, further expanding the cells with strong fluorescence signals after 6-10 days (6 days, 7 days, 8 days, 9 days, 10 days and the like), and finally screening the RAW264.7 single cell stable cell line transfected with the red fluorescent protein.

As a preferred technical scheme of the invention, the screening method of the RAW264.7 single cell stable cell line transfected with the red fluorescent protein specifically comprises the following steps:

(1) RAW264.7 cells were resuspended in complete medium and (1-10). times.10⁴Spreading the cells/hole on a cell culture plate for culturing for 20-30 h; replacing the complete culture medium with a serum-free basal medium, adding a serum-free basal medium containing pDSRED2-C1 plasmid and transfection reagent, and performing transfection;

(2) after 20-25h of transfection, the culture medium in the step (1) is changed into a G418 screening culture medium, wherein the G418 screening culture medium is a complete culture medium containing 800-1000 mu G/mL G418;

(3) after 10-15 days, selecting the cells in the transfection positive step (2) for digestion according to the fluorescence observation result, suspending in 400-activated 500 mu G/mL G418 screening culture medium, diluting the cells, and then paving in a 96-well plate for single cell dilution and paving;

(4) and screening single cell wells, culturing and replacing the culture medium with a G418 screening culture medium, expanding the cells with strong fluorescence signals after 6-10 days, culturing and replacing the culture medium with the G418 screening culture medium, further expanding the cells with strong fluorescence signals after 6-10 days, and finally screening the RAW264.7 single cell stable cell line transfected with the red fluorescent protein.

In a second aspect, the invention provides a RAW264.7 single-cell stable cell line transfected with red fluorescent protein, which is obtained by screening the method for screening the RAW264.7 single-cell stable cell line transfected with red fluorescent protein.

Compared with the prior art, the invention has the following beneficial effects:

Drawings

FIG. 1 is a diagram showing the state of cells cultured under different pressures for 1 week in example 1;

FIG. 2 is a diagram showing the state of cells cultured under different pressures for 2 weeks in example 1;

FIG. 3 is a graph showing a visible light field (100X) in example 2;

FIG. 4 is a graph showing fluorescence visual field (100X) in example 2;

FIG. 5 is a graph showing the visible field (100X) and the fluorescent field (100X, exposure 1.5s) of the cells obtained in example 2 after passage 1 time under the pressure condition of 1000. mu.g/mLG 418 selection medium;

FIG. 6 is a graph showing the visible field (100X) and the fluorescent field (100X, exposure 1.5s) of the cells obtained in example 2 after passage 2 under the pressure condition of 1000. mu.g/mLG 418 selection medium;

FIG. 7 is a visible field image (100X) and a fluorescent field image (100X, exposure time 300ms) of the cells obtained in example 3 passaged 1 time under the pressure condition of 1000. mu.g/mLG 418 selection medium;

FIG. 8 is a graph of a visible field (100X) and a fluorescent field (100X, exposure time 300ms) of cells obtained in example 3 after passage 2 under a pressure condition of 1000. mu.g/mLG 418 of the selection medium;

FIG. 9 is a visible field view (100X) and a fluorescent field view (100X, exposure time 300ms) of cells obtained in example 3 passaged 3 times under a pressure condition of 1000. mu.g/mLG 418 screening medium;

FIG. 10 is a visible field view (100X) and a fluorescent field view (100X, exposure time 300ms) of the cells obtained in example 3 after passage 4 times under the pressure condition of 1000. mu.g/mLG 418 screening medium;

FIG. 11 is a graph of a visible field (100X) and a fluorescent field (100X, exposure time 300ms) of the cells obtained in example 4 passaged 1 time under a pressure condition of 1000. mu.g/mLG 418 selection medium;

FIG. 12 is a graph showing a visible field (100X) and a fluorescent field (100X, exposure time 300ms) of the cells obtained in example 4 after passage 2 under the pressure condition of 1000. mu.g/mLG 418 selection medium;

FIG. 13 is a visible field view (100X) and a fluorescent field view (100X, exposure time 300ms) of the cells obtained in example 4 after passage 3 times under the pressure condition of 1000. mu.g/mLG 418 screening medium;

FIG. 14 is a visible field view (100X) and a fluorescent field view (100X, exposure 300ms) of the cells obtained in example 4 passaged 4 times under the pressure condition of 1000. mu.g/mLG 418 screening medium.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The following examples relate to materials and equipment including the following:

example 1

This example was used to determine the optimal screening dose for G418 as follows:

(1) p5 generation cells (density: 2X 10) of RAW264.7 in logarithmic growth phase were collected⁵individuals/mL, viability: 96.5%) at 5X 10⁴One/well of complete medium without G418 (DMEM + 10% FBS) was plated in 1 24-well plate and placed in CO₂Incubation in an incubator;

(2) preparing 1mg/mL of G418 mother liquor, adding the G418 mother liquor into a cell culture medium (DMEM + 10% FBS), and preparing 7 groups of G418 screening culture media with different concentrations, namely 300, 400, 500, 600, 700, 800 and 900 mu G/mL;

(3) after 24 hours of incubation of the 24-well plate, using a G418-free complete culture medium (Control) and a 7-group G418 screening culture medium for liquid exchange, continuously exchanging liquid with the corresponding culture medium every 48 hours for 2 weeks (liquid exchange is carried out for 3 times), and observing the cell state and the growth condition by using a microscope in the process;

(4) according to the growth condition of the cells under the action of different G418 screening concentrations within 2 weeks, selecting the experimental condition that the cells die gradually within 2 weeks, and determining the cell density and the G418 screening concentration used in the formal transfection experiment. The dose was chosen to gradually kill the cells within 2 weeks, i.e. the dose was chosen to kill the cells within 1 week and completely kill the cells within 2 weeks.

The results are shown in FIGS. 1 and 2 (in FIG. 1, a, b, c, and d are graphs showing the state of cells cultured at 300. mu.g/mL, 700. mu.g/mL, 800. mu.g/mL, and 900. mu.g/mL, respectively, for 1 week; and in FIG. 2, a, b, c, and d are graphs showing the state of cells cultured at 300. mu.g/mL, 700. mu.g/mL, 800. mu.g/mL, and 900. mu.g/mL, respectively, for 2 weeks), from which: when the concentration of G418 is at least about 700 mug/mL, the cells can be killed after 1 week of action, and the cells can be completely killed after 2 weeks of action when the concentration of G418 reaches at least 800 mug/mL. Based on the above experimental results, we determined that the G418 concentration is greater than or equal to 800. mu.g/mL during the transfection of RAW264.7 cells with the red fluorescent protein DsRed.

Example 2

This example provides a method for screening RAW264.7 single cell stable cell line transfected with red fluorescent protein, comprising the following steps:

(1) preparing a plasmid pDsRED2-C1, confirming that a red fluorescent protein DsRed sequence is correct through sequencing, and preparing the plasmid with the concentration of 377 ng/mu L;

(2) preparing 1mg/mL of G418 mother liquor, and adding the G418 mother liquor into a complete culture medium (DMEM + 10% FBS) to prepare a screening culture medium of 500 mu G/mL and 1000 mu G/mL;

(3) RAW264.7 cells were resuspended in complete medium and at 5X 10⁴Spreading the cells/hole in a 24-hole cell culture plate for culturing for 24 hours; the complete medium was replaced with serum-free basal medium, to which was added serum-free basal medium containing pDSRED2-C1 plasmid and transfection reagent (1. mu.g plasmid + 2. mu.l transfection reagent: (1. mu.g plasmid + 2. mu.l)

HD transfection reagent) +100 μ L DMEM) for transfection; placing the transfected cells in an incubator for continuous culture, and replacing the cells with a complete culture medium after 4 hours;

(4) 24h after transfection, the culture medium in the step (2) is changed to a G418 screening culture medium, and the G418 screening culture medium is a complete culture medium containing 1000 mug/mL G418;

(5) after 14 days, according to the fluorescence observation result (exposure time 300ms, excitation wavelength 510-560nm, emission wavelength 590nm), selecting the cells with the most obvious red fluorescence signal in the step (2), digesting, suspending in G418 screening culture medium of 500 mug/mL, diluting the cells, and then spreading in a 96-well plate for single cell dilution and plating; among them, the visible light field (100X) of the cell in which the red fluorescence signal is most pronounced is shown in FIG. 3, and the fluorescence field (100X) is shown in FIG. 4;

(6) and screening single cell wells, culturing and replacing with 500 mu G/mL G418 screening culture medium, expanding the cells with strong fluorescence signals to a 24-well plate after 7 days, culturing and replacing with 500 mu G/mL G418 screening culture medium, further expanding the cells with strong fluorescence signals to a 6-well plate after 7 days, and finally screening the RAW264.7 single cell stable cell line transfected with the red fluorescent protein.

Example 3

(2) preparing 1mg/mL of G418 mother liquor, and adding the G418 mother liquor into a complete culture medium (DMEM + 10% FBS) to prepare a screening culture medium of 400 mu G/mL and 800 mu G/mL;

(3) RAW264.7 cells were resuspended in complete medium and at 5X 10⁴Spreading the cells/hole in a 24-hole cell culture plate for culturing for 22 h; the complete medium was replaced with serum-free basal medium, to which was added serum-free basal medium containing pDSRED2-C1 plasmid and transfection reagent (1. mu.g plasmid + 2. mu.l transfection reagent: (1. mu.g plasmid + 2. mu.l)

HD transfection reagent) +100 μ L DMEM) for transfection; the transfected cells are put into an incubator for continuous cultureCulturing for 4h, and then changing into a complete culture medium;

(4) 26h after transfection, the culture medium in the step (2) is changed to a G418 screening culture medium, and the G418 screening culture medium is a complete culture medium containing 800 mug/mL G418;

(5) after 15 days, according to the fluorescence observation result (exposure time 300ms, excitation wavelength 510-560nm, emission wavelength 590nm), selecting the cells with the most obvious red fluorescence signal in the step (2), digesting, suspending in a G418 screening culture medium of 400 mu G/mL, diluting the cells, and then paving in a 96-well plate for single cell dilution and paving;

(6) and screening single cell wells, culturing and replacing with 400 mu G/mL G418 screening culture medium, expanding the cells with strong fluorescence signals to a 24-well plate after 7 days, culturing and replacing with 400 mu G/mL G418 screening culture medium, further expanding the cells with strong fluorescence signals to a 6-well plate after 7 days, and finally screening the RAW264.7 single cell stable cell line transfected with the red fluorescent protein.

Example 4

(2) preparing 1mg/mL of G418 mother liquor, and adding the G418 mother liquor into a complete culture medium (DMEM + 10% FBS) to prepare screening culture media of 450 mu G/mL and 900 mu G/mL;

(3) RAW264.7 cells were resuspended in complete medium and at 5X 10⁴Spreading the cells/well in a 24-well cell culture plate for culturing for 26 h; the complete medium was replaced with serum-free basal medium, to which was added serum-free basal medium containing pDSRED2-C1 plasmid and transfection reagent (1. mu.g plasmid + 2. mu.l transfection reagent: (1. mu.g plasmid + 2. mu.l)

(4) 22h after transfection, the culture medium in the step (2) is changed to a G418 screening culture medium, and the G418 screening culture medium is a complete culture medium containing 900 mu G/mL G418;

(5)12 days later, according to the fluorescence observation result (exposure time 300ms, excitation wavelength 510-560nm, emission wavelength 590nm), selecting the cells with the most obvious red fluorescence signal in the step (2), digesting, suspending in a G418 screening culture medium with 450 mu G/mL, diluting the cells, and then paving in a 96-well plate for single cell dilution and paving;

(6) and screening single cell wells, culturing and replacing with 450 mu G/mL G418 screening culture medium, expanding the cells with strong fluorescence signals to a 24-well plate after 7 days, culturing and replacing with 450 mu G/mL G418 screening culture medium, further expanding the cells with strong fluorescence signals to a 6-well plate after 7 days, and finally screening the RAW264.7 single cell stable cell line transfected with the red fluorescent protein.

Example 5

In this example, the passaging stability of the RAW264.7 single-cell stable cell line obtained in example 2 was evaluated, and the cells were passaged 2 times under a pressure condition of 1000. mu.g/mLG 418 selection medium, and fluorescence detection was performed, and the visible light field pattern (100X) and the fluorescence field pattern (100X, exposure 1.5s) at the 1 st generation were as shown in FIG. 5 (the former and latter correspond to a and b in this order), and the visible light field pattern (100X) and the fluorescence field pattern (100X, exposure 1.5s) at the 2 nd generation were as shown in FIG. 6 (the former and latter correspond to a and b in this order).

As can be seen from the figure: under the pressure of 1000. mu.g/mL, the insertion of the red fluorescent protein fragment is relatively stable, and the fluorescent signal is not reduced obviously after 1 and 2 passages.

Example 6

In this example, the passage stability of the RAW264.7 single-cell stable cell line obtained in example 3 was evaluated, and the cells were passaged 4 times under a pressure condition of 1000. mu.g/mLG 418 selection medium, and fluorescence detection was performed, and the visible light field pattern (100 ×) and the fluorescence field pattern (100 ×, exposure time 300ms) of the 1 st passage are shown in FIG. 7 (the former and latter correspond to a and b, respectively); the visible field pattern (100 ×) and the fluorescence field pattern (100 ×, exposure 300ms) of the generation 2 are shown in FIG. 8 (the former and latter correspond to a and b, respectively); the visible light field pattern (100 ×) and the fluorescence field pattern (100 ×, exposure 300ms) of the 3 rd generation are shown in fig. 9 (the former and latter correspond to a and b, respectively); the visible light field pattern (100 ×) and the fluorescent field pattern (100 ×, exposure time 300ms) of the 4 th generation are shown in fig. 10 (the former and latter correspond to a and b, respectively).

As can be seen from the figure: under the pressure of 1000 mug/mL, the insertion of the red fluorescent protein fragment is relatively stable, and the reduction of the fluorescent signal after 1-4 passages is not obvious.

Example 7

In this example, the passage stability of the RAW264.7 single-cell stable cell line obtained in example 4 was evaluated, and the cells were passaged 4 times under a pressure condition of 1000. mu.g/mLG 418 selection medium, and fluorescence detection was performed, and the visible light field pattern (100 ×) and the fluorescence field pattern (100 ×, exposure time 300ms) of the 1 st passage are shown in FIG. 11 (the former and latter correspond to a and b, respectively); the visible field pattern (100 ×) and the fluorescence field pattern (100 ×, exposure 300ms) of the 2 nd generation are shown in FIG. 12 (the former and latter correspond to a and b, respectively); the visible light field pattern (100 ×) and the fluorescence field pattern (100 ×, exposure 300ms) of the 3 rd generation are shown in fig. 13 (the former and latter correspond to a and b, respectively); the visible light field pattern (100 ×) and the fluorescent field pattern (100 ×, exposure 300ms) of the 4 th generation are shown in fig. 14 (the former and latter correspond to a and b, respectively).

In conclusion, the method for screening the RAW264.7 single cell stable cell line transfected with the red fluorescent protein can obtain the RAW264.7 single cell line with good transfection effect and has good passage stability.

The applicant states that the present invention is illustrated by the above examples, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. A method for screening a RAW264.7 single cell stable cell line transfected with a red fluorescent protein, wherein the method for screening comprises the following steps:

(2) applying a screening pressure to the cells obtained in step (1);

2. The method for screening a RAW264.7 single cell stable cell line transfected with red fluorescent protein according to claim 1, wherein the operation method of step (1) comprises: resuspending RAW264.7 cells in complete medium and plating in cell culture plate for culture; the complete medium was replaced with a serum-free basal medium, to which the pDsRED2-C1 plasmid and transfection reagents were added for transfection.

3. The method of claim 2, wherein the method comprises selecting a stable cell line from RAW264.7 single cells transfected with red fluorescent proteinThe plating density of the RAW264.7 cells is (1-10) x 10⁴One/hole, preferably 5X 10⁴Per well;

preferably, the culturing time is 20-30 h.

4. The method for screening a RAW264.7 single cell stable cell line transfected with red fluorescent protein according to claim 2 or 3 wherein said serum-free basal medium is a serum-free DMEM medium;

preferably, the pDSRED2-C1 plasmid is dissolved in serum-free DMEM medium at a concentration of 8-12 mg/L;

preferably, the transfection reagent is dissolved in serum-free DMEM medium and has a volume percentage of 1-3%.

5. The method for screening a RAW264.7 single cell stable cell line transfected with red fluorescent protein according to any of claims 1-4 wherein said applying a screening pressure to cells means screening the cells with G418;

preferably, the concentration of G418 in the G418 screening medium is 800-.

6. The method for screening a RAW264.7 single cell stable cell line transfected with red fluorescent protein according to any of claims 1-5, wherein said applying a screening pressure to the cells is performed 20-25h after transfection.

7. The method for screening RAW264.7 single cell stable cell line transfected with red fluorescent protein according to any of claims 1-6 wherein said fluorescence observation is performed 10-15 days after the application of screening pressure;

preferably, the resuspension refers to resuspending the cells in 400-500. mu.g/mL G418 screening medium;

8. The method for screening a RAW264.7 single cell stable cell line transfected with red fluorescent protein according to any of claims 1-7, wherein said method for screening a single cell stable cell line comprises: and screening single cell wells, culturing and replacing the culture medium with a G418 screening culture medium, expanding the cells with strong fluorescence signals after 6-10 days, culturing and replacing the culture medium with the G418 screening culture medium, further expanding the cells with strong fluorescence signals after 6-10 days, and finally screening the RAW264.7 single cell stable cell line transfected with the red fluorescent protein.

9. The method for screening a RAW264.7 single cell stable cell line transfected with red fluorescent protein according to any of claims 1-8, wherein the screening method comprises the following steps:

10. The method for screening RAW264.7 single cell stable cell line transfected with red fluorescent protein according to any of claims 1-8, wherein the obtained RAW264.7 single cell stable cell line transfected with red fluorescent protein is screened.