CN116678810A - Cell Proliferation Immunofluorescence Detection Kit and Its Application - Google Patents

Abstract

The invention provides a cell proliferation immunofluorescence detection kit and application thereof. Based on the existing nucleic acid fluorescent labeling method, the present invention effectively distinguishes G2 and M phase cells by introducing a special marker for M phase cells (phosphorylated histone H3). By selecting and optimizing the basic conditions of the experiment, the basic nucleic acid markers, newly synthesized nucleic acid markers, and special markers of M phase cells are compatible, so as to complete the comprehensive detection of the four cell cycles. Further combined with immunofluorescence cytometry to qualitatively or quantitatively observe the subcellular structure of cells in different cell proliferation cycles, so as to accurately reflect all the information of the cell proliferation cycle. The cell cycle detection method provided by the invention has the advantages of simple operation, high efficiency, sensitivity, cheap and easy-to-obtain reagents, etc., can perform qualitative, quantitative and location detection on cell samples, and has broad application prospects.

Description

Cell Proliferation Immunofluorescence Detection Kit and Its Application

technical field

The invention relates to the field of biotechnology, in particular to a cell proliferation immunofluorescence detection kit and its application.

Background technique

Cell proliferation is the basis for maintaining life and is a very complex and finely regulated physiological process. According to the characteristics of nucleic acid, protein and overall cell changes during the proliferation process, the cell proliferation cycle can be artificially divided into four cycles: G1, S, G2 and M.

The occurrence and development of many genetic diseases, infectious diseases, tumors and other diseases are directly related to abnormal cell proliferation cycle. Therefore, the detection of cell proliferation cycle is an important means to judge the pathological mechanism of cells, and it is also a commonly used experimental method in clinical and basic biomedical research. .

Immunofluorescence combined with fluorescence microscope and automatic data processing system can qualitatively or quantitatively observe the number, proportion, subcellular structure and expression distribution of related proteins in different cycle cells, and is one of the most commonly used technical means in the field of biomedicine.

At present, there are mainly two types of detection methods for the cell proliferation cycle at home and abroad. The first type is to directly use nucleic acid reagents (such as PI, DAPI, HOECHST, etc.) analyze. This method is very simple and easy to operate; however, the analysis of experimental results is very difficult and prone to many errors, especially the analysis of cells in S and M phases is very unreliable. At present, it is only suitable for some drug screening combined with flow cytometry.

The second type is based on the first type of nucleic acid reagents, and additionally uses nucleotide analogs (such as EdU) to label newly synthesized DNA for distinguishing S phase cells. Since cells in S phase can be amplified and displayed by marker signals, this method can divide cells into three cycles of G1, S and G2M. This method can be used to study some pathological processes that affect the G1/S, S/G2 cycle, but there is no way to detect the G2/M cycle. Not only can it not provide all the information on the cell proliferation cycle, but also the diseases that affect the G2/M cycle transition cannot be tested at all. Therefore, there is an urgent need to establish a method for effectively distinguishing and identifying cell structures in M and G2 phases.

Contents of the invention

The purpose of the present invention is to provide a cell proliferation immunofluorescence detection kit and its application.

In order to achieve the purpose of the present invention, in a first aspect, the present invention provides the use of phosphorylated histone H3 as a marker of M phase in the cell proliferation cycle.

In a second aspect, the present invention provides an application of a reagent for detecting phosphorylated histone H3 in the preparation of a detection reagent for distinguishing the M phase of the cell proliferation cycle.

For the aforementioned application, the reagent for detecting phosphorylated histone H3 is an anti-phosphorylated histone H3 antibody, such as an anti-human p-H3-Alexa Fluro647 antibody (Abcam, ab237418).

In a third aspect, the present invention provides a cell proliferation immunofluorescence detection kit (cell cycle fluorescence detection kit), the kit comprising phosphorylated histone H3 antibody.

Further, the kit also includes at least one of a newly synthesized DNA marker, a cell fixative, a membrane breaking agent, and a fluorescent staining agent for total nucleic acid.

The newly synthesized DNA marker may be Edu (5-ethynyl-2-deoxyuridine, a substitute for thymine).

The cell fixative may be 1-4% (preferably 2%) paraformaldehyde. Its configuration method is as follows: dissolving powder paraformaldehyde in double distilled water to prepare mother liquor with a mass ratio of 40%. Heat and stir and add NaOH dropwise until the powder is completely dissolved, and freeze the mother solution at -20°C. When using, take the mother solution and dissolve it, and dilute the mother solution to 1-4v/v% working solution with PBS.

The membrane breaking agent may be PBS containing 0.1-1% (preferably 0.1%) Triton.

The fluorescent stain can be DAPI.

In the fourth aspect, the present invention provides the application of the kit in detecting the M phase of the cell proliferation cycle (including non-disease diagnosis and treatment purposes).

In a fifth aspect, the present invention provides a method for detecting the M phase of the cell proliferation cycle (including non-disease diagnosis and treatment purposes), comprising the following steps:

(1) Cells are placed in a complete cell culture medium containing 1-10 μM Edu for 0.5-4 hours;

(2) After the culture is over, wash the cells with PBS 2-3 times, fix the cells with 2% paraformaldehyde at room temperature for 20 minutes, wash the cells with washing solution (PBS containing 10% FCS) (remove the fixing solution) ;

(3) After treating the cells with a membrane-breaking agent (PBS containing 0.1v/v% Triton) at room temperature for 20 minutes, wash the cells with a washing solution (remove the membrane-breaking agent);

(4) Resuspend the cells with Edu labeling solvent (100mM Tris solution containing 1mM CuSO ₄ , 0.1μM azide-Alexa Fluro 488 and 100mM ascorbic acid), treat the cells at room temperature for 20 minutes, and wash the cells with washing solution (to remove excess reagents) ;

(5) Add 10 μg/ml p-H3-Alexa Fluro647 to resuspend the cells, stain the cells at 4°C for 30 minutes, and wash the cells with washing solution (to remove excess antibodies);

(6) Adjust the cells to an appropriate concentration with PBS, and spin the cell suspension onto a glass slide;

(7) After staining with 1 μg/ml DAPI at room temperature for 10 minutes, wash with PBS 4-5 times;

(8) Add sample buffer solution (90v/v% glycerol+10v/v%pH9 PBS) dropwise, add a cover slip, and mount the slide;

(9) Observing the cell sample under a fluorescence microscope.

If the experimental object is adherent cells, step (6) needs to be removed, and the cells are directly cultured on the glass slide in step (2).

The cells are mammalian somatic cells, preferably primate somatic cells, more preferably human somatic cells.

By virtue of the above technical solutions, the present invention has at least the following advantages and beneficial effects:

In order to break through the limitation of traditional cell proliferation cycle detection technology, the present invention provides a novel cell cycle fluorescence detection kit. Based on the existing nucleic acid fluorescent labeling method, the present invention effectively distinguishes G2 and M phase cells by introducing a special marker for M phase cells (phosphorylated histone H3). By selecting and optimizing the basic conditions of the experiment, the basic nucleic acid markers, newly synthesized nucleic acid markers, and special markers of M phase cells are compatible, so as to complete the comprehensive detection of the four cell cycles. Further combined with immunofluorescence cytometry to qualitatively or quantitatively observe the subcellular structure of cells in different cell proliferation cycles, so as to accurately reflect all the information of the cell proliferation cycle. The cell cycle detection method provided by the invention has the advantages of simple operation, high efficiency, sensitivity, cheap and easy-to-obtain reagents, etc., can perform qualitative, quantitative and location detection on cell samples, and has broad application prospects.

Description of drawings

Fig. 1 is the result of detecting the proliferation cycle of K562 cells using the cell cycle fluorescence detection kit in a preferred embodiment of the present invention.

Fig. 2 is the flow cytometry detection verification result of stained cells in a preferred embodiment of the present invention.

Fig. 3 is the result of the screening optimization experiment of fixative membrane breaker in the preferred embodiment of the present invention; Wherein, A: 2% paraformaldehyde and saponin (saponin); B: 2% paraformaldehyde and containing 0.1v/v% Triton's PBS; C: low temperature ethanol; D: commercial kit (ThermoFisher Scientific, Cat. No. A10266).

Figure 4 is the preferred experimental results of the p-H3-Alexa Fluro647 antibody in a preferred embodiment of the present invention; among them, A: pS28-H3-Histone-APC (Miltenyi, 130-126-509); B: pS28-H3-Histone- Alexa Fluro647 (BD, 558609); C: pS10-H3-Histone-Alexa Fluro647 (Abcam, ab5176); D: pS28-H3-Histone-Alexa Fluro647 (Abcam, ab237418).

Figure 5 is the preferred experimental results of Edu from different sources in the preferred embodiment of the present invention; among them, A: Invitrogen, A10266; B: Aladdin, E131265; C: RHAWN, R014330; D: MCE, HY118411.

Detailed ways

The present invention aims to provide M-phase cell-specific marker protein for distinguishing G2 and M-phase cells, so as to comprehensively detect the subcellular structure of cells in four different cell proliferation cycles. In addition, by selecting suitable newly synthesized DNA and basic DNA markers, it can be used to mark nucleic acids in different cell cycles; Compatible with proteins, etc.

The present invention adopts following technical scheme:

Through extensive research, the inventors have found that phosphorylated histone H3 is only expressed in M phase cells, but not expressed in other cell cycles, thus it can become an excellent M phase marker. Through protein sequence analysis, several candidate antibodies were screened. Combining flow cytometry and immunofluorescence to determine the best antibody and prepare a specific fluorescent antibody for detecting the expression of phosphorylated histone H3. Second, select sensitive and specific Edu as nucleic acid analogs to label newly synthesized DNA, and label with corresponding fluorescent markers; select DAPI to label DNA, which has wide adaptability and can label nucleic acids in a variety of reagent environments. Finally, among many commonly used combinations of fixative-membrane breaking agent-fluorescent staining agent, the best combination of reagents was successfully screened out, which is compatible with all the reagents used in this experiment, so that the four cycles of subcellular structures can be It was observed qualitatively, quantitatively and localized under a fluorescence microscope.

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

The percentage sign "%" involved in the present invention refers to the mass percentage unless otherwise specified; but the percentage of the solution refers to the grams of solute contained in 100 mL of the solution, unless otherwise specified.

Reagents and instruments used in the following examples:

Anti-human p-H3-Alexa Fluro647 antibody was purchased from Abcam (Cat. No. ab237418)

Azide-Alexa Fluro488 was purchased from ThermoFisher Scientific (Cat. No. A10266). K562 cells were purchased from ATCC (CCL-243).

FCS was purchased from Gibco (16000044).

Flow cytometer was purchased from BD FACSAria (BD, 643181).

Laser confocal microscope was Leica TCS SP5.

Example 1 Immunofluorescence detection of cell cycle (differentiation of M and G2 phase cells)

1. K562 cells were cultured with 1-10 μM Edu (purchased from Aladdin, E131265); 1-10 mM Edu working solution was added to the complete cell culture medium (RPMI 1640 medium containing 10% fetal bovine serum, purchased from ThermoFisher, Cat. No. 11875093), cultured for 0.5-4 hours according to the cell proliferation rate.

2. Harvest the cells; after washing 2-3 times with low-temperature PBS, resuspend and fix the cells with 2% paraformaldehyde at room temperature for 20 minutes, then wash with washing solution (PBS+10v/v% FCS) to remove the fixing solution

3. After treating the cells with a membrane-breaking agent (PBS+0.1v/v% Triton) at room temperature for 20 minutes, wash with a washing solution to remove the membrane-breaking agent.

4. Resuspend the cells with Edu labeling solvent mixture (100mM Tris mixed with 1mM CuSO4, 0.1μM azide-alexaFluro 488 and 100mM ascorbic acid solution), treat the cells at room temperature for 20 minutes, and wash the cells with washing solution to remove excess reagents.

5. Add 10 μg/ml p-H3-Alexa Fluro647 to resuspend the cells. After staining the cells at 4°C for 30 minutes, wash them thoroughly with washing solution to remove excess antibodies.

6. Adjust the cells to a concentration of 0.1×10 ⁶ /ml to 1×10 ⁶ /ml with PBS, take 100 μl of the cell suspension and spin it onto a glass slide.

7. After staining with 1 μg/ml DAPI at room temperature for 10 minutes, wash thoroughly with PBS 4-5 times.

8. Add sample buffer solution (90v/v% glycerol + 10v/v% pH9 PBS) dropwise, add a cover slip, and mount the slide.

9. Observe the cell sample under a fluorescence microscope.

The above experimental method is based on suspension cells as an example. If the subject of the experiment is adherent cells, the cells can be cultured on a glass slide and then transferred to a petri dish. The basic process is similar, and the process of cell flake removal can be omitted.

The detection results of the proliferation cycle of K562 cells using the cell cycle fluorescence detection kit are shown in Figure 1. Among them, the upper left panel shows that DAPI stains the DNA of all cells. The fluorescence intensity after staining varies among different cell cycle cells; the upper right panel shows newly synthesized DNA stained by Edu, and these positively stained cells represent S-phase cells; the lower left panel shows phosphorylated histone H3 antibody-stained cells, these cells are Cells in M phase, whose chromosomes are characteristic.

The stained cells were verified by flow cytometry and the results are shown in Figure 2. Among them, the upper left image is a scatter plot, showing the size and intracellular status of all cells; the upper right image shows the distribution of individual cells; the lower left image is divided into three types of proliferation: G1, S and G2/M after staining with DAPI and Edu-AF488 Cycle; the lower right panel shows that p-H3-Alexa Fluro647 can distinguish G2/M phase cells, and the antibody positive cells are M phase cells. The staining efficiency-sensitive specificity in Figure 1 was demonstrated.

Embodiment 2 Screening optimization experiment of fixative membrane breaking agent

The experimental process is basically the same as in Example 1, only the combination of fixative and membrane breaker in steps 2 and 3 is changed: A: 2% paraformaldehyde and saponin (saponin); B: 2% paraformaldehyde and 0.1v /v% Triton in PBS; C: low temperature ethanol; D: commercial kit (ThermoFisher Scientific, Cat. No. A10266). The experimental results are shown in Figure 3. A-D results are from the above four combinations respectively. P3 in the figure shows cells in G2/M phase. Among them, the result obtained by combination B is similar to that of the existing commercial two-reagent combination, and is the preferred applied reagent.

Example 3 p-H3-Alexa Fluro647 antibody optimization experiment

The experimental process is basically the same as in Example 1, only the antibody in step 5 is changed, and p-H3-Alexa Fluro647 antibody from different sources is selected to compare and select the best stained antibody A: pS28-H3-Histone-APC (Miltenyi, 130- 126-509); B: pS28-H3-Histone-Alexa Fluro647 (BD, 558609); C: pS10-H3-Histone-AlexaFluro647 (Abcam, ab5176); D: pS28-H3-Histone-Alexa Fluro647 (Abcam, ab237418 ). The verification results are shown in Figure 4 with flow cytometry histogram. A-D results are from the above four antibody staining results respectively. Panel P4 shows antibody-stained cells in M phase. Among them, the result obtained by combination D is obviously better than that of the other three groups, and it is the best applied reagent.

Example 4 Different sources of Edu optimization experiment

The experimental process is basically the same as in Example 1, except that the Edu in step 1 is changed, and Edu from different sources is selected for comparison to select the best dyeing result. A: Invitrogen, A10266; B: Aladdin, E131265; C: RHAWN, R014330; D: MCE, HY118411. The verification results are shown in Figure 5 with the flow cytometry scatter plot. A-D results are from the above four reagents respectively. Although Edu in the four experimental groups can be well embedded in S phase cells, and the proportion of positive cells is basically the same. Considering factors such as product purity, price and supply location, Edu produced by Aladdin is finally preferred.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. Use of phosphorylated histone H3 as a marker of M phase in the cell proliferation cycle.

2. Use of a reagent for detecting phosphorylated histone H3 in the preparation of a detection reagent for distinguishing M phase of a cell proliferation cycle.

3. The use according to claim 2, wherein the reagent for detecting phosphorylated histone H3 is an anti-phosphorylated histone H3 antibody.

4. The use according to claim 2, wherein the reagent for detecting phosphorylated histone H3 is an anti-human p-H3-Alexa f luro647 antibody.

5.A cell proliferation immunofluorescence assay kit, characterized in that the kit comprises a phosphorylated histone H3 antibody.

6. The kit of claim 5, further comprising at least one of a newly synthesized DNA marker, a cell fixative, a membrane breaker, and a nucleic acid fluorescent stain.

7. The kit of claim 6, wherein the phosphorylated histone H3 antibody is an anti-human p-H3-Alexa f luro647 antibody; and/or

The newly synthesized DNA marker is Edu; and/or

The cell fixative is 1-4% paraformaldehyde; and/or

The membrane breaker is PBS containing 0.1-1v/v% Triton; and/or

The nucleic acid fluorescent stain is DAPI.

8. Use of the kit according to any one of claims 5-7 for detecting the M phase of the cell proliferation cycle for non-disease diagnostic and therapeutic purposes.

9. A method for detecting the M phase of a cell proliferation cycle comprising the steps of:

(1) Culturing the cells in a cell complete medium containing 1-10 mu M Edu for 0.5-4 hours;

(2) After the culture is finished, washing the cells with PBS for 2-3 times, fixing the cells with 2% paraformaldehyde at room temperature for 20 minutes, and then washing the cells with a washing solution;

wherein the washing solution is PBS containing 10% FCS;

(3) After treating cells with a membrane breaker at room temperature for 20 minutes, washing the cells with a washing solution;

wherein the membrane breaker is PBS containing 0.1v/v% Triton;

(4) Re-suspending the cells with Edu labeling solvent, treating the cells at room temperature for 20 minutes, and washing the cells with a washing solution;

wherein the Edu labeling solvent comprises 1mM CuSO ₄ 100mM Tris solution of 0.1. Mu.M azide-Alexa Fluro488 and 100mM ascorbic acid;

(5) Adding 10 mug/ml of anti-human p-H3-Alexa Fluro647 antibody to re-suspend the cells, staining the cells at 4 ℃ for 30 minutes, and washing the cells with a washing solution;

(6) The cells are regulated to proper concentration by PBS, and the cell suspension is thrown onto a glass slide;

(7) After staining with 1. Mu.g/ml DAPI for 10 minutes at room temperature, washing with PBS 4-5 times;

(8) Dropping sample buffer solution, adding cover glass, and sealing;

(9) Observing the cell sample under a fluorescence microscope;

the methods are for non-disease diagnosis and treatment purposes.

10. The method according to claim 9, wherein the cells are mammalian somatic cells, preferably primate somatic cells, more preferably human somatic cells.