Simple and rapid cell adhesion method and application
By simultaneously using poly L-lysine and paraformaldehyde coating methods on solid phase substrates, the contradiction between structural protection and detection effect during suspension cell adhesion and fixation is solved, and the stability of cell adhesion and detection flexibility is improved.
Patent Information
- Application Number
- CN202411990747.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-05-09
AI Technical Summary
The prior art requires the use of paraformaldehyde to fix it when adhering suspended cells to the surface of solid phase matrix, resulting in cell structure protection but antibody binding is blocked, affecting the detection effect, and increasing operational complexity and time.
The method of coating the solid phase substrate by simultaneously adding polyL-lysine and paraformaldehyde to enhance cell adhesion, and by simplifying steps, the fixation of paraformaldehyde is reduced or eliminated, maintaining the original state of protein localization in the cell.
The stable adhesion of cells on solid phase substrates is achieved, the risk of cell shedding is reduced, the original state of proteins within the cell is retained to the greatest extent, the detection flexibility and free selection are improved, and experimental time is saved.
Smart Images
Figure CN119959536A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and specifically relates to a simple, rapid and universal new method for cell adhesion to a solid matrix surface, which can be used for characteristic analysis such as immunofluorescence staining of suspended cells, enzymatic reaction of cells in situ, and cellular localization and expression of bioactive substances. Background Art
[0002] In the field of biomedicine, detecting the in situ distribution and abundance of intracellular biomolecules is a common and important experimental method. Immunofluorescence staining is an important method for detecting the localization of intracellular proteins. To this end, the cells must first be attached to the surface of a solid substrate such as a slice or a culture dish before subsequent staining. There are currently two common methods for adhering suspended cells to a solid substrate. One is to centrifuge the cells using a centrifuge, and the other is to achieve attachment by cross-linking with poly-L-lysine. Lysine can be polymerized to any desired chain length, and poly-L-lysine binds to most solid supports through its charged side chains. The positively charged polymer will provide binding sites for the cells that carry a negative charge as a whole, thereby achieving cell adhesion on the solid substrate.
[0003] Usually, after the suspended cells are attached to the solid substrate by the above two methods, they are fixed with organic solvents (such as ethanol or acetone) or cross-linking reagents (such as 4% paraformaldehyde), and then subsequent biological experiments such as immunofluorescence staining are carried out. Paraformaldehyde can better maintain the cell structure than organic solvents, so it is currently the most commonly used fixation method. Paraformaldehyde can chemically cross-link the free amino groups in the cell. When different molecules are cross-linked, a network structure is formed, connecting the various structural components of the cell to achieve the purpose of cross-linking fixation. This cross-linking effect can protect the cell structure, but it may also hinder the binding of some antibodies to the free amino groups in the cell, or cause the conformation of some antigen components to change due to the cross-linking effect, thereby greatly weakening the binding of some antibodies to antigens, and ultimately leading to a decrease in the antigen detection rate. For example, it has been reported in the literature that the fixation of paraformaldehyde will affect the detection of green fluorescent protein (GFP), causing the conformation of GFP protein to change and unable to produce fluorescence. On the other hand, for proteins located on the cell surface, paraformaldehyde fixation is not required, but in the subsequent primary and secondary antibody incubation process, multiple changes in the liquid will cause cell shedding and affect the staining effect. If paraformaldehyde is added for fixation to prevent falling off, it will increase additional operations and waiting time.
[0004] While increasing the matrix binding force, reducing the cell fixation strength as much as possible can increase the detection rate of the target molecule. Therefore, finding a method to increase the attachment of cells to the substrate while avoiding the intracellular paraformaldehyde fixation effect as much as possible is an urgent problem to be solved.
[0005] The method of the present invention can quickly and easily enhance the adhesion of cells on a solid substrate, making the adhesion more stable and less likely to fall off than by cross-linking with poly-L-lysine alone, while reducing or even eliminating unnecessary fixation of paraformaldehyde in the cells, thereby retaining the original state of protein localization inside the cells to the greatest extent, and improving the flexibility and free selectivity of detectable intracellular proteins. Summary of the invention
[0006] The purpose of the present invention is to provide a simple, rapid and universal cell adhesion method, which can be used for characteristic analysis such as immunofluorescence staining of suspended cells, enzymatic reaction of in situ cells and cell localization and expression of bioactive substances. The present invention only uses a method of coating a solid phase substrate by adding poly-L-lysine and paraformaldehyde at the same time, which is more efficient, rapid and convenient, and only takes about half an hour from cell collection to cell attachment.
[0007] In order to achieve the above object, the present invention provides a simple and rapid cell adhesion method, which comprises the following steps:
[0008] Step S1. Mix the poly-L-lysine solution and the paraformaldehyde solution and add them to the solid phase container, and incubate at room temperature for coating;
[0009] Step S2. discard the solution in the solid phase vessel and wash with distilled water;
[0010] Step S3. Centrifuging the suspended cells and then resuspending them in a buffer solution;
[0011] Step S4. Centrifuge the cells resuspended in step S3 and resuspend them in a buffer solution;
[0012] Step S5. Add the cells resuspended in step S4 to the solid phase vessel coated in step S2, and incubate at room temperature to allow the cells to adhere to the solid phase vessel.
[0013] According to a preferred embodiment of the present invention, in step S1, in the mixed solution formed by mixing the poly-L-lysine solution and the paraformaldehyde solution, the final concentration of poly-L-lysine is 0.4-0.6 mg / mL, and the final concentration of paraformaldehyde is 1.5-2.5%.
[0014] In actual operation, a poly-L-lysine solution with a concentration of 0.8 to 1.2 mg / mL and a poly-formaldehyde solution with a concentration of 3 to 5% can be selected, and then the two can be mixed in equal volumes.
[0015] In the present invention, the solid phase vessel includes but is not limited to solid phase matrices such as glass slides, well plates, culture dishes or culture bottles.
[0016] According to a preferred embodiment of the present invention, in step S1, the purpose of incubation at room temperature is to allow poly-L-lysine and paraformaldehyde to fully coat the solid phase vessel. Preferably, the incubation time at room temperature is 10 to 20 minutes.
[0017] According to the present invention, in step S2, the purpose of washing with distilled water is to wash away the residual solution. Excessive washing times will affect the coating, so it is better to wash once.
[0018] According to the present invention, the suspended cells need to be resuspended twice. In step S3 and step S4, the centrifugal conditions can be the same or different, and are independently: a rotation speed of 300-500g and a time of 4-6 minutes.
[0019] The present invention has no particular limitation on the buffer solution, and it can be selected conventionally in the art. In a specific embodiment, the buffer solution used in steps S3 and S4 is a phosphate buffer solution (PBS).
[0020] The present invention has no special requirements for the first resuspension, but in the second resuspension (i.e., step S4), the cells are preferably suspended at 10 5 ~5×10 5 Resuspend cells / mL in buffer.
[0021] According to a preferred embodiment of the present invention, in step S5, the incubation is used to make the cells adhere to the solid phase vessel. Preferably, the incubation time is 8 to 12 minutes.
[0022] The method of the present invention is applicable to various cell types. In one example, the cells used are human monocytes (THP-1 cells).
[0023] After using the cell adhesion method of the present invention, more cells are more firmly attached to the solid phase vessel, which can be subsequently used for suspended cell immunofluorescence staining, in situ cell enzymatic reaction monitoring, or cell localization and expression characteristic analysis of bioactive substances.
[0024] The beneficial effects of the present invention include:
[0025] 1. Enhance cell adhesion on solid substrates through a quick and simple method. Compared with the traditional adhesion effect through single poly-L-lysine cross-linking, the method of the present invention makes cell adhesion more stable, thereby avoiding or reducing the cell shedding phenomenon caused by the liquid replacement and incubation process required for subsequent immunofluorescence experiments.
[0026] 2. Reduce or even eliminate unnecessary fixation within the cell, thereby preserving the original state of protein localization within the cell to the greatest extent. By avoiding the polyformaldehyde fixation of the entire cell, the adverse consequences such as reduced reactivity of bioactive substances and detection distortion caused by formaldehyde cross-linking are reduced, and the flexibility and freedom of choice of detectable intracellular proteins are improved.
[0027] 3. The method of the present invention is fast and simple. Compared with the traditional method of first coating with poly-L-lysine and then fixing with paraformaldehyde after cell attachment, the present invention can make cells firmly attached to the substrate after co-coating with poly-L-lysine and paraformaldehyde, thereby saving experimental time (it only takes about half an hour from cell collection to cell attachment) and improving work efficiency.
[0028] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.
[0030] Figure 1 The bright field photographic images of the attached cells in each embodiment of the present invention and the comparative example under a light microscope. Scale bar: 100 micrometers. In the figure, PFA is 4% paraformaldehyde, and poly-L-Lysine is poly-L-lysine.
[0031] Figure 2 The images taken under a fluorescence microscope after DAPI staining in Comparative Example 1 of the present invention are shown. The images of different fields of view under 4x and 10x microscopes are respectively selected for display. The scale of the field of view under 4x microscope is 500 microns; the scale of the field of view under 10x microscope is 100 microns.
[0032] Figure 3 The photographic images taken under a fluorescence microscope after DAPI staining in Example 1 of the present invention are shown in Figure 1. The images of different fields of view under 4x and 10x microscopes are selected for display. The scale of the field of view under 4x microscope is 500 microns; the scale of the field of view under 10x microscope is 100 microns.
[0033] Figure 4 The photographic images taken under a fluorescence microscope after DAPI staining in Comparative Example 2 of the present invention are shown in Figure 2. The images of different fields of view under a 4x microscope and a 10x microscope are selected for display. The scale of the field of view under a 4x microscope is 500 microns; the scale of the field of view under a 10x microscope is 100 microns.
[0034] Figure 5The photographic images taken under a fluorescence microscope after DAPI staining in Comparative Example 3 of the present invention are shown. The images of different fields of view under a 4x microscope and a 10x microscope are respectively selected for display. The field of view scale of the 4x microscope is 500 microns; the field of view scale of the 10x microscope is 100 microns. DETAILED DESCRIPTION
[0035] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.
[0036] In the following examples and comparative examples, the reagent components used are as follows:
[0037]
[0038] The cells used in the following examples and comparative examples are human monocytes (THP-1 cells).
[0039] Example 1
[0040] This example is used to illustrate a cell adhesion method provided by the present invention.
[0041] S1. Coating of well plates: Add 0.5 mL of 1 mg / mL poly-L-lysine and 0.5 mL of 4% paraformaldehyde to a 12-well plate; mix well and let stand at room temperature for 15 minutes.
[0042] S2. After coating, discard the solution in the well plate and wash it once with distilled water.
[0043] S3. Centrifuge the suspended cells at 400 g for 5 minutes and then resuspend them in PBS.
[0044] S4, the cells resuspended in step S3 were centrifuged again at 400g for 5 minutes, and then the cells were centrifuged at 10 5 cells / mL resuspended in PBS;
[0045] S5. Add the cells resuspended in step S4 to the well plate coated in step S2 and incubate at room temperature for 10 minutes.
[0046] S6. Observe the cells under a light microscope and take photos in the bright field.
[0047] S7. Discard the cell supernatant, add membrane permeabilization solution along the wall, and let it stand at room temperature for 5 minutes.
[0048] S8. Discard the permeabilization solution, add DAPI staining solution, and observe and take pictures under a fluorescence microscope after 3 minutes.
[0049] Comparative Example 1
[0050] Cell adhesion was performed according to the method of Example 1, except that only poly-L-lysine was used to coat the well plate.
[0051] That is, step S1 includes:
[0052] Add 1 mL of 1 mg / mL poly-L-lysine to the well plate and let it stand at room temperature for 15 minutes.
[0053] Comparative Example 2
[0054] Cell adhesion was performed according to the method of Example 1, except that the well plate was first coated with poly-L-lysine and then with paraformaldehyde.
[0055] That is, step S1 includes:
[0056] 1 mL of 1 mg / mL poly-L-lysine was added to the well plate and allowed to stand at room temperature for 15 minutes. The liquid was then discarded and 1 mL of 4% paraformaldehyde was added and allowed to stand at room temperature for 15 minutes.
[0057] Comparative Example 3
[0058] Cell adhesion was performed according to the method of Example 1, except that the well plate was first coated with paraformaldehyde and then with poly-L-lysine.
[0059] That is, step S1 includes:
[0060] 1 mL of 4% paraformaldehyde was added to the well plate, and the well plate was allowed to stand at room temperature for 15 minutes. The liquid was then discarded, and 1 mL of 1 mg / mL poly-L-lysine was added, and the well plate was allowed to stand at room temperature for 15 minutes.
[0061] Experimental Results
[0062] The results of bright field photography and fluorescence microscopy are as follows Figure 1 , Figure 2 , Figure 3 , Figure 4 and Figure 5 shown.
[0063] Figure 1 These are bright field photographic images of cells attached in various embodiments and comparative examples of the present invention under a light microscope. Figure 1 It shows that the adhesion effects of the cells in Comparative Example 1, Example 1, Comparative Example 2 and Comparative Example 3 after cell adhesion are basically the same.
[0064] Figure 2-5 The cells in Comparative Example 1 and Example 1, Comparative Example 2 and Comparative Example 3 were treated with membrane permeabilization solution and photographed under a fluorescence microscope after adding DAPI staining solution.
[0065] Figure 2 It was shown that in Comparative Example 1, after being treated with the membrane-breaking solution, a considerable portion of cells fell off and cell agglomeration occurred.
[0066] Figure 3 It is shown that in Example 1, after being treated with the membrane-breaking solution, the cells did not show obvious shedding and the distribution was still relatively uniform.
[0067] Figure 4 It is shown that in Comparative Example 2, after being treated with the membrane-breaking solution, the cells showed slight shedding and aggregation phenomena, and the degree of shedding was lighter than that in Comparative Example 1, but heavier than that in Example 1.
[0068] Figure 5 It is shown that in Comparative Example 3, after being treated with the membrane-breaking solution, the cells partially detached and aggregated, and the degree of detachment was comparable to that of Comparative Example 1.
[0069] It can be seen from the above experimental results that after being treated with the membrane-breaking solution, the cells in Example 1 have the best attachment effect, the least detached cells, and the best retention of cell integrity.
[0070] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.
Claims
1. A simple and rapid cell adhesion method, characterized in that: The cell adhesion method comprises the following steps: Step S1. Mix the poly-L-lysine solution and the paraformaldehyde solution and add them to the solid phase container, and incubate at room temperature for coating; Step S2. discard the solution in the solid phase vessel and wash with distilled water; Step S3. Centrifuging the suspended cells and then resuspending them in a buffer solution; Step S4. Centrifuge the cells resuspended in step S3 and resuspend them in a buffer solution; Step S5. Add the cells resuspended in step S4 to the solid phase vessel coated in step S2, and incubate at room temperature to allow the cells to adhere to the solid phase vessel.
2. The cell adhesion method according to claim 1, characterized in that: In step S1, in the mixed solution formed by mixing the poly-L-lysine solution and the paraformaldehyde solution, the final concentration of the poly-L-lysine is 0.4-0.6 mg / mL, and the final concentration of the paraformaldehyde is 1.5-2.5%.
3. The cell adhesion method according to claim 1, characterized in that: In step S1, the solid phase vessel is a glass slide, a well plate, a culture dish or a culture bottle.
4. The cell adhesion method according to claim 1, characterized in that: In step S1, the incubation time at room temperature is 10 to 20 minutes.
5. The cell adhesion method according to claim 1, characterized in that: In step S2, the number of washing is one time.
6. The cell adhesion method according to claim 1, characterized in that: In steps S3 and S4, the centrifugal conditions are independently: a rotation speed of 300 to 500 g and a time of 4 to 6 minutes.
7. The cell adhesion method according to claim 1, characterized in that: In steps S3 and S4, the buffer solution is a phosphate buffer solution.
8. The cell adhesion method according to claim 1, characterized in that: In step S4, the cells were divided into 10 5 ~5×10 5 Resuspend cells / mL in buffer.
9. The cell adhesion method according to claim 1, characterized in that: In step S5, the incubation time is 8 to 12 minutes.
10. Use of the cell adhesion method according to any one of claims 1 to 9 in immunofluorescence staining of suspended cells, monitoring of enzymatic reactions of cells in situ, or analysis of cell localization and expression characteristics of bioactive substances.