CN113376079A - Method for analyzing transfection efficiency using CountStar Rigel System in combination with cell staining - Google Patents
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Abstract
The invention discloses a method for analyzing transfection efficiency by using a CountStar Rigel system in combination with cell staining, which comprises the following steps: 1) cell staining of the transfected cell samples using CloneDetect reagent; the CloneDetect reagent can be specifically combined with proteins produced by cells and secreted to the cell surface; 2) detecting the cell sample stained in the step 1) by using a CountStar Rigel system, and reading a FITC signal to obtain the transfection efficiency of the transfected cell sample. The method has the characteristics of quickly, simply and accurately obtaining the result of the transfection efficiency, can overcome the defects of time and labor waste and difficulty in subsequent cutting in the fusion protein method, has better accuracy than the GFP cell method, and has the advantages of simplified operation and improved efficiency compared with a fluorescence microscope or a flow cytometer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting transfection efficiency of mammalian cell plasmid DNA by using a CountStar Rigel system in combination with cell staining.
Background
Currently, the method of analyzing transfection efficiency is usually to use a fluorescent protein to label or indicate the target protein. This method can be classified into the following two methods. The first method is a fusion protein method, in which a fluorescent protein and a target protein are expressed as a fusion protein, and the expression level of the fluorescent protein is directly related to the expression level of the target protein. However, in the expression and production of pharmaceutical proteins, it is not permissible for the pharmaceutical protein to carry a fluorescent protein, since the subsequent cleavage is time-consuming, laborious and difficult, which adversely affects the quality and cost of the pharmaceutical protein. The second method is GFP cell-based comparison, which is to express fluorescent protein and target protein separately and independently according to the same transfection method, and then to compare the ratio of fluorescent protein to the transfection efficiency of target protein. This type of approach is an indirect approach, since the fluorescent protein is usually not the same size and structure as the target protein, and thus the transfection efficiency is different, and the drawback of directly comparing the two is that it causes a large error.
The prior art solution that can be used to detect the fluorescent signal of cells is to use fluorescence microscopy or flow cytometry. The fluorescence microscope uses ultraviolet rays as a light source to irradiate an object to be detected, so that the object to be detected emits fluorescence, and then the shape and the position of the object to be detected are observed under the microscope. It is used for researching the absorption and transportation of substances in cells, the distribution and positioning of chemical substances and the like. These substances may be those which fluoresce when irradiated with ultraviolet rays, or those which do not fluoresce but fluoresce when irradiated with ultraviolet rays after being stained with a fluorescent dye or a fluorescent antibody. The fluorescent microscope has the defects of manual adjustment during observation, extremely low efficiency and large manpower requirement. Fluid cytometers are devices for the automated analysis and sorting of cells which rapidly measure, store and display a series of important biophysical, biochemical characteristic parameters of dispersed cells suspended in a liquid, and from which a given cell subpopulation can be sorted according to a preselected parameter range. In the flow cytometry, fluorescence generated by a cell stained by fluorescence after being excited by a suitable light is measured by converting the fluorescence into an electrical signal by a photoelectric converter. Flow cytometers are carefully adjusted before and even during use to ensure reliability and optimization of operation. The flow cytometer has the disadvantages of high price, complex operation, high difficulty in handling, professional training of operators and great adverse effect on experimental results due to non-professional operation.
CountStar Rigel is a novel automatic instrument designed by ALIT Life Sciences, is based on image detection, is matched with multiple fluorescence channels, and carries out quantitative analysis by collecting cell information in an image. The fluorescence microscopic imaging and the analysis of the statistical population are integrated, not only can provide statistical data of the cell population, but also can obtain an image of a single cell, thereby providing morphological information of the cell, and simultaneously displaying cell images and analysis results of cell viability, apoptosis, transfection, period and the like on a display screen. The system is specifically optimized for analysis of primary cells in peripheral blood, including PBMCs, CAR-T, NK cells and MSCs commonly used in cell therapy. The automatic control system is high in automation degree and simple and convenient to operate, results can be known immediately by only one key, and the price is obviously lower than that of a flow cytometer. The cell staining method currently used by CountStar Rigel for transfection efficiency is still the two methods described above, namely the fusion protein method and the GFP cell-specific method.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a novel method for analyzing the transfection efficiency, which has the characteristics of quickly, simply and accurately detecting and obtaining the result of the transfection efficiency, can overcome the defects of time and labor waste and difficulty in subsequent cutting in a fusion protein method, has better accuracy than a GFP cell comparison method, and has the advantages of simplified operation, convenient use and efficiency improvement compared with a fluorescence microscope or a flow cytometer.
In order to solve the technical problems, the invention provides the following technical scheme:
a method for analyzing transfection efficiency using the CountStar Rigel system in conjunction with cell staining, the method comprising the steps of:
1) cell staining of the transfected cell samples using CloneDetect reagent; the CloneDetect reagent can be specifically combined with a protein produced by cells and secreted to the surfaces of the cells, and the CloneDetect reagent has a fluorescent group;
2) detecting the cell sample stained in the step 1) by using a CountStar Rigel system, and reading a FITC signal to obtain the transfection efficiency of the transfected cell sample.
Specifically, in the step 1), the specific binding means that the CloneDetect reagent is specifically bound to the Fc terminal or the light chain terminal of a drug protein produced by cells and secreted to the cell surface.
Specifically, in the step 1), the CloneDetect reagent is an antibody protein.
Specifically, in the step 1), the fluorescent group is a fluorescein isothiocyanate group.
Specifically, in the step 1), the method for staining the cells is as follows: resuspending the transfected cell sample with CloneDetect reagent and incubating at room temperature for 20-60 min; and then washing the cells for 1-3 times by using a permeabilization buffer solution, and then washing the cells for 1-3 times by using a PBS buffer solution to obtain a cell sample which is dyed.
Preferably, the obtained cell sample after complete staining is resuspended in PBS buffer, preserved at 0-4 ℃ and used for detection by CountStar Rigel system.
Preferably, the storage time does not exceed 1 hour.
Specifically, in the step 2), a sample of the stained cells is sampled and added to the well of the slide of the CountStar Rigel system, and the "GFP transfection efficiency" mode using the CountStar Rigel system is selected for reading the FITC signal.
Specifically, in the step 2), the transfection efficiency results are displayed on the screen of the CountStar Rigel system.
In particular, the method is used to characterize the expression of a drug protein in a cell sample.
The invention develops a new method for analyzing the transfection efficiency by combining a CountStar Rigel system with a CloneDetect reagent cell staining technology, and the method can quickly, simply and accurately detect and obtain the result of the transfection efficiency.
In the two cell staining methods for detecting the transfection efficiency in the prior art, the fusion protein is difficult to cut, or the error is large, and the transfection efficiency of the target protein to the host cell cannot be directly measured, and the transfection efficiency of the target protein to the host cell is deduced by the transfection efficiency of the fusion protein or GFP protein in an indirect mode.
In the prior art, the commonly used fluorescence microscope has low cost and low efficiency and needs a large amount of manpower. The fluid cytometer has high cost, complex operation and high training requirement on operators, and many laboratories have no use condition. Therefore, both approaches have great limitations for more efficient development of stable cell lines, shortening of the development cycle of biopharmaceuticals, and reduction of the development cost of biopharmaceuticals.
CloneDetect reagent was used in the method of the present invention to stain transfected cells. The reagent is an antibody protein which can be specifically combined with the Fc end or the light chain end of a drug protein, and a fluorescent group, such as a FITC compound (fluorescein isothiocyanate) which can emit light, is connected with the antibody protein. The FITC signal emitted by the fluorophore can be detected and read. If the cell sample produces a drug protein and is secreted to the cell surface, the drug protein will be specifically bound by the CloneDetect reagent, thereby causing the drug protein on the cell surface to produce a FITC signal. While no signal is detectable by cells that do not produce the drug protein. Transfection efficiency can be obtained by calculating the proportion of cells with signal. The production of the CloneDetect reagent is animal-origin-free and meets the safety requirements of pharmaceutical production on the reagent. The agent is only loosely contacted with the drug protein and the cell itself for a short period of time and does not require additional cleavage after purification. The specific combination of the reagent and the drug protein is that after the drug protein is expressed, a small part of sample is taken and analyzed by the reagent, the dyeing is a non-covalent bond connection and is reversible, and the reagent can be dissociated by changing the substrate concentration and other conditions. More importantly, the drug protein was not stained by the reagent except for the sample. The fusion protein method is to express the fusion protein and the drug protein at the same time, and permanently connect them by covalent bond, and can only cut by consuming energy after the final drug purification, such as using hydrolytic enzyme. This is one of the significant advantages of the method of the invention over the fusion protein method.
The CloneDetect reagent involved in the invention is designed by Molecular Devices and is used for the own ClonePix systemTMAnd reagents of the CloneMedia system to improve workflow productivity for screening and selecting mammalian cell lines. The following is an introduction to the range of applications of the product: 1. selection of a detection agent specific for a secreted mouse, rat or human IgG; 2. when detecting antigen-specific secretion of hybridomas, complex starter factor products can be used; 3. the provided tissue culture was verified to be sterile and azide-free; 4. for ready-to-use "spray" applications in atomizer spray heads. In general, the CloneDetect reagent, which is generally used to measure protein secretion in single cells for the purpose of screening high expressing cell clones, rather than to measure transfection efficiency, has never been used in the CountStar novel instrument.
The method provided by the invention provides a scheme for combining a CountStar Rigel system with a CloneDetect reagent, can overcome the defects of time and labor consumption of subsequent cutting in a fusion protein method, has better accuracy than a GFP cell method, has the advantages of high automation degree, no need of manually adjusting focal length and high detection efficiency compared with a fluorescence microscope, and has the advantages of low cost, simplicity in operation, convenience in use and efficiency improvement compared with a flow cytometer.
Drawings
FIG. 1 shows the fluorescence signal from the CountStar Rigel system of example 1.
FIG. 2 shows the results of the analysis of CountStar Rigel system in example 1 using FCS Expression De Novo software.
FIG. 3 shows the results of the CountStar Rigel system of test 1 in example 2.
FIG. 4 shows the results of the transfection efficiency measured by the flow cytometer of test 1 in example 2.
FIG. 5 shows the results of the CountStar Rigel system of test 2 in example 2, analyzed using FCS Expression De Novo software.
FIG. 6 shows the results of the transfection efficiency measured by the flow cytometer of test 2 in example 2.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example one
1. Cell staining
Reagent:
reagent 1.CloneDetect (Molecular device, product No. K8295 or K8205)
Reagent 2. intracellular fixing and permeabilizing buffer kit (thermo fisher scientific, product No. 88-8824-00)
Reagent 3 PBS buffer (thermo fisher scientific, product No. 10010023)
The method comprises the following steps:
A. cell staining process
1. 1 × E6 cells were collected in a 1.7ml centrifuge tube.
2. Spin at 350G for 5 min and remove the supernatant.
3. Wash with 0.7ml PBS and mix gently by vortexing. The supernatant was discarded.
4. Add 500. mu.L of IC fixation buffer in reagent 2 and mix with pulsed vortex to fix the cells.
5. Incubate at room temperature for 20-60 minutes. And (4) avoiding light.
6. 0.7mL of one-fold permeation buffer was added and centrifuged at 400x g for 5 minutes at room temperature. The supernatant was discarded.
7. And 6, repeating the step.
8. Buffer permeabilized with one fold in reagent 2 at 1: CloneDetect was diluted at a ratio of 200.
9. The cell pellet was resuspended in 100. mu.L of diluted CloneDetect.
10. Incubate at room temperature for 20-60 minutes. And (4) avoiding light.
11. One-fold permeation buffer in 0.7ml reagent 2 was added and centrifuged at 400x g for 5 minutes at room temperature. The supernatant was discarded.
12. Step 11 is repeated. Mix gently by vortexing.
13. Wash twice with 1ml PBS buffer and mix gently by vortexing.
14. The stained cells were resuspended in an appropriate volume of PBS buffer.
15. The samples were stored in a refrigerator at 4 degrees celsius until the signal was read. Not more than 1 hour.
2. Signal reading with CountStar
Description of the apparatus: CountStar Rigel is a novel image-based automated cellular analysis platform with a full-spectrum fluorescence microscope, FCS Expression De Novo software, which can analyze and report data and images. A plurality of fluorescence channels are provided, different fluorescence can be analyzed, and dyes with different wavelengths can be supported. The method is simple and convenient to operate, user-friendly, free of manual focal length adjustment and high in stability and accuracy.
Step (ii) of
1. Take 20. mu.l of surface stained cells to wells of a CountStar slide.
2. The "GFP transfection efficiency" mode of CountStar Rigel was chosen for reading FITC signal.
3. The location of the sample well is selected.
4. The sample name and cell type are entered.
5. The corresponding fluorescence channel is selected. Adjust 'gain' and exposure time.
6. Clicking the "start" button
7. The results of the transfection rate read are displayed on the screen, the results are shown in FIG. 1, and the instrument automatically gives a result that 94% of the cells have fluorescent signals. The raw data can also be exported and analyzed by FCS De Novo software results are shown in FIG. 2, the left panel in FIG. 2 shows all morphologically healthy and intact cells in black boxes, and the right panel shows that 94.28% of these healthy cells show GFP + with a fluorescence signal, i.e., 94.28% transfection efficiency.
The above process basically does not need manual debugging after the detection parameters are selected, and the detection efficiency is high. The detection is carried out according to the flow, the detection of a transfected cell sample can be completed in only 3 minutes, and the detection of 20 samples per hour can be completed on average when a large number of samples are used. While the prior flow cytometer combined with the fusion protein method/GFP cell comparison method only takes 2 minutes to run samples, the cleaning and debugging steps are generally required to be started for 30 minutes additionally before each use, a cleaning step of 30 minutes is additionally required after the use is finished, and 20 samples are detected in the same way, and the total time is 1 hour and 40 minutes.
Example 2
The method of the invention has a detection accuracy level that is substantially consistent with flow cytometry detection. Flow cytometry can become a detection method generally accepted at present, and is based on the advantage of accuracy. The method of the invention finds that the experimental result is very close to that of the flow cytometer by comparison. The settings for the comparative experiments were as follows:
test 1: the samples were transfected HEK293 cells. Sampling 1, and analyzing the transfection efficiency by adopting a CountStar Rigel system provided by the invention and combining a GFP cell type ratio method; and 2, sampling, namely detecting the transfection efficiency by combining the conventional flow cytometry with a GFP cell comparison method, wherein the operation flow is carried out according to the operation recommended by equipment manufacturers. The results of the detection are shown in FIGS. 3 and 4.
In FIG. 3, sample 1 shows a GFP + data of 38.68% at CountStar; in FIG. 4, sample 2 showed 32.3% GFP + data on a loss-of-cell cytometer. Considering the systematic error of the two instruments themselves, we consider the two data to be very close in accuracy.
Test 2: the samples were transfected CHO cells. Sampling 3, and analyzing the transfection efficiency by adopting a CountStar Rigel system provided by the invention and combining a cell staining method; and 4, sampling, and detecting the transfection efficiency by adopting the conventional flow cytometry combined with a cell staining method, wherein the operation flow is carried out according to the operation recommended by equipment manufacturers. The detection results are shown in fig. 5 and 6.
In FIG. 5, sample 3 showed 12.04% CHO cell transfection efficiency after CountStar output data was treated with FCS De Novo software; in FIG. 6, sample 4 showed 10.2% CHO cell transfection efficiency on a flow cytometer. Considering the systematic error of the two instruments themselves, we consider these two data to be very close in accuracy.
The above two examples were tested with two different types of cells, respectively, where the only variable controlled in the test was the instrument difference, and the test results both showed similar results for the method of the present invention and the flow cytometer, indicating that both reached very similar levels of detection accuracy. And the method of the invention has significant advantages in detection efficiency and cost input compared with the flow cytometry.
In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for analyzing transfection efficiency using the CountStar Rigel system in conjunction with cell staining, comprising the steps of:
1) cell staining of the transfected cell samples using CloneDetect reagent; the CloneDetect reagent can be specifically combined with a protein produced by cells and secreted to the surfaces of the cells, and the CloneDetect reagent has a fluorescent group;
2) detecting the cell sample stained in the step 1) by using a CountStar Rigel system, and reading a FITC signal to obtain the transfection efficiency of the transfected cell sample.
2. The method of claim 1, wherein in step 1), the specific binding is that the CloneDetect reagent specifically binds to the Fc terminus or the light chain terminus of a drug protein produced by cells and secreted to the cell surface.
3. The method of claim 2, wherein in step 1), the CloneDetect reagent is an antibody protein.
4. The method of claim 1, wherein in step 1), the fluorophore is a fluorescein isothiocyanate group.
5. The method of claim 1, wherein in step 1), the method of staining the cells is: resuspending the transfected cell sample with CloneDetect reagent and incubating at room temperature for 20-60 min; and then washing the cells for 1-3 times by using a permeabilization buffer solution, and then washing the cells for 1-3 times by using a PBS buffer solution to obtain a cell sample which is dyed.
6. The method of claim 5, wherein the stained cell sample is resuspended in PBS buffer and stored at 0-4 ℃ for detection by the CountStar Rigel system.
7. The method of claim 6, wherein the holding time is no more than 1 hour.
8. The method of claim 1, wherein in step 2), a sample of stained cells is sampled and applied to a well of a slide of a CountStar Rigel system, and the reading of the FITC signal is selected using the "GFP transfection efficiency" mode of the CountStar Rigel system.
9. The method of claim 8, wherein in step 2), the transfection efficiency results are displayed on a screen of a CountStar Rigel system.
10. The method of any one of claims 1 to 9, wherein the method is used to characterise the expression of a pharmaceutical protein in a cell sample.
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