CN117214438A - Cell five-period detection kit and application - Google Patents
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Abstract
The invention provides a cell five-period detection kit and application thereof. According to the invention, the G2 and M-phase cells are distinguished by introducing the M-phase cell special marker (phosphorylated histone H3), and the comprehensive detection of five cell cycles is realized by combining the G0-phase cell detection technology. And cell proliferation cycle of the suspension cells is detected by further optimizing and adjusting cell labeling reagents and conditions in combination with flow cytometry. The invention carries out iterative upgrade on the basis of the existing four-cycle cell proliferation cycle detection kit, and can realize the comprehensive detection of five cell cycles. Compared with the four-cycle cell proliferation cycle detection kit, the EdU is used for replacing the BrdU doping labeling method, so that the BrdU antibody is not required to be used, and the detection is simple and quick.
Description
Technical Field
The invention relates to the field of cell biology, in particular to a cell five-cycle detection kit and application thereof.
Background
Cell proliferation is the basis for life support, and is a very complex and precisely regulated physiological process. Dividing the proliferation cycle of the cells into four cycles of G1, S, G and M according to the overall change characteristics of nucleic acid, protein and cells in the proliferation process; cells that are in a resting state and that have not been put into the proliferation cycle are referred to as G0 phase cells.
The research shows that the occurrence and development of many genetic diseases, infectious diseases, tumors and other diseases are directly related to abnormal cell proliferation cycle, so that the detection of the cell proliferation cycle is an important means for judging the pathological mechanism of cells, and is also a common experimental method in clinical and scientific research work.
At present, two types of detection methods for cell proliferation cycles at home and abroad exist, the first type is to directly dye cell DNA with nucleic acid reagents (such as PI, DAPI, hoechst and the like), and analyze the cell cycle according to different cell cycle cell DNA contents. The method is simple to operate, but the experimental result is difficult to analyze, more errors are easy to generate, and particularly, the analysis on S and M-phase cells is very unreliable. At present, the method is only suitable for the primary screening of some pharmaceutical biological effects.
The second category is to label newly synthesized DNA with nucleotide analogs (e.g., edU/Brdu) based on the first category of nucleic acid reagents to distinguish S phase cells. Since the S-phase cells can be displayed by the amplification of the marker signal, the method can distinguish between the three periods of G1, S and G2M. The method can be used for researching some pathological processes affecting the G1/S, S/G2 cycle, but the pathological processes cannot be detected for the G2/M cycle, so that the whole information of the cell proliferation cycle is difficult to provide, and diseases affecting the G2/M cycle transition cannot be detected at all. Meanwhile, since the detection of the G0 phase is not introduced, all cells cannot be detected comprehensively. Therefore, there is a need to develop a new cell proliferation cycle detection method to reflect all information of the cell proliferation cycle comprehensively and accurately.
Disclosure of Invention
The invention aims to provide a cell cycle detection kit capable of comprehensively detecting five cell cycles (G0, G1, S, G2 and M) and application thereof.
The invention is characterized in that: previous studies showed that phosphorylated histone H3 is expressed only in M-phase cells and not in other cycles, so it was used as M-phase cell marker. Further studies have found that specific staining agents (PY) bind selectively to RNA in the cytoplasm, whereas once cells enter the G1 phase of the cell proliferation cycle from the G0 phase, their intracellular RNA content will increase significantly, thereby allowing differentiation of low PY stained cells into G0 phase cells. According to the invention, the G2 and M-phase cells are distinguished by introducing the M-phase cell special marker, and the comprehensive detection of five cell cycles is realized by combining the G0-phase cell detection technology. And cell proliferation cycle of the suspension cells is detected by further optimizing and adjusting cell labeling reagents and conditions in combination with flow cytometry.
To achieve the object of the present invention, in a first aspect, the present invention provides a five-cycle cell detection kit, which comprises a phosphorylated histone H3 detection reagent, a nucleic acid fluorescent dye, an EdU solution (aladine-E131265), an Alexa fluor 488-Azide working solution, and the like.
The phosphorylated histone H3 assay reagent may be an anti-phosphorylated histone H3 antibody, preferably p-H3-Alexa fluor 647 (Cell Signalling Tech, 3458S).
The nucleic acid fluorescent dye may be selected from DAPI, PY, etc.
The working concentration of the EdU solution was 10-50. Mu.M.
Wherein, the substance which is subjected to covalent reaction with EdU under the catalysis of sodium citrate is Alexa Fluro 488-Azide (AAT-1701).
DAPI, PY can recognize the binding cell DNA, RNA, respectively, relatively specifically and distinguish G0 phase from other cell cycles due to their staining intensity associated with different cell cycles.
The Alexa Fluro 488-Azide working solution contains 1mM CuSO 4 2. Mu.M of Azide-Alexa Fluro 488 and 100mM sodium ascorbate in 100mM Tris.
Further, the kit also comprises a cell fixative, a cell membrane breaker and the like.
The cell fixative is 2% -4% paraformaldehyde.
The cell rupture agent is PBS containing 0.1% -0.5% Triton X-100.
In a second aspect, the invention provides the use of the kit in cell cycle assays (including non-diagnostic purposes).
In a third aspect, the invention provides a method of cell cycle detection (including non-diagnostic purposes), the method comprising:
(1) Inoculating cells into a cell culture medium containing Edu for culture;
(2) Harvesting the cells, washing, and fixing the cells with a cell fixative;
(3) The fixed cells are treated with a cell rupture agent;
(4) Mixing the cells subjected to membrane rupture treatment with Alexa Fluro 488-Azide working solution for reaction;
(5) Washing the harvested cells and incubating with a phosphorylated histone H3 assay reagent;
(6) Washing and re-suspending the cells, adding nucleic acid fluorescent dye (DAPI, PY) for staining, washing the cells after staining, and carrying out flow cytometry after re-suspending the cells;
wherein, the cell fixative, the cell rupture agent, the phosphorylated histone H3 detection reagent, the Alexa Fluro 488-Azide working solution and the nucleic acid fluorescent dye can be from the above kit.
Further, the method comprises the steps of:
1) Inoculating the cells into a cell culture medium with the EdU concentration of 10-50 mu M for culturing for 0.5-4 hours;
2) After harvesting the cells, washing with PBS, fixing the cells with 4% paraformaldehyde at room temperature for 10-30 minutes (preferably fixing the cells with 4% paraformaldehyde at room temperature for 15 minutes), washing with a washing solution to remove paraformaldehyde;
3) The fixed cells are treated with a cell membrane breaker at room temperature for 10-30 minutes (preferably 15 minutes), and then washed with a washing solution to remove the membrane breaker;
4) Adding Alexa Fluro 488-Azide working solution into the cells after membrane rupture treatment, staining the cells for 20-30 minutes (preferably 30 minutes) at room temperature, and washing the cells with a washing solution;
5) Resuspending the cells in a wash solution, mixing the cells with a phosphorylated histone H3 assay reagent (final concentration of p-H3-Alexa Fluro 647 is 1-10. Mu.g/mL, preferably 1. Mu.g/mL), incubating for 20-30 minutes (preferably 20 minutes) at normal temperature, washing the cells with the wash solution;
6) Cells were resuspended in wash solution, DAPI and PY were added sequentially to final concentrations of 0.1-2. Mu.g/mL and 0.1-1. Mu.g/mL, respectively, and stained at room temperature for 10-20 minutes (preferably, DAPI was 2. Mu.g/mL, PY was 0.4. Mu.g/mL, and stained at room temperature for 10 minutes), then washed and resuspended in PBS for flow cytometry.
The wash solution used in steps 2) to 5) was PBS containing 10% FCS or FBS. The pH of the PBS used was 7.2-7.4.
In the present invention, the cells are mammalian somatic cells.
Compared with the existing cell proliferation cycle detection kit product, the invention has at least the following advantages and beneficial effects:
the invention uses the M-phase cell marker for cell cycle detection to effectively distinguish G2 and M-phase cells, realizes the comprehensive detection of four cell proliferation cycles, and can accurately reflect all information of the cell proliferation cycles.
And secondly, the invention uses the G0 phase cell marker-Pyronine Y (PY) for cell cycle detection for the first time, integrates with the detection of four cell proliferation cycles of G1, S, G2 and M, and realizes the comprehensive detection of five cell cycles.
The cell cycle detection method provided by the invention has the advantages of simplicity in operation, high efficiency, low price and easiness in obtaining reagents and the like, can be used for detecting large-scale cell samples, and has a cost performance far higher than that of similar products sold in the market, and a wide application prospect.
The invention carries out iterative upgrade on the basis of the existing four-cycle cell proliferation cycle detection kit, and can realize the comprehensive detection of five cell cycles. Compared with the four-cycle cell proliferation cycle detection kit, the EdU is used for replacing the BrdU doping labeling method, so that the BrdU antibody is not required to be used, and the detection is simple and quick.
Drawings
FIG. 1 shows the results of a five cycle cell proliferation cycle assay for PBMC using a five cycle cell assay kit according to a preferred embodiment of the present invention. Wherein, the left graph shows PY to divide the mixed cells into two parts of G0 phase and cell proliferation phase (including G1, S, G and M phase), the middle graph shows EdU and DAPI to divide the mixed cells into G1, S and G2M phase cells, and the right graph shows phosphorylated histone H3 to divide the G2M phase cells.
FIG. 2 shows the results of detection of the proliferation cycle of PBMC cells using the first nucleic acid reagent DAPI-labeled three-cycle kit according to the preferred embodiment of the present invention.
FIG. 3 shows the results of a three-cycle kit labeled with a second nucleotide analogue for detecting the proliferation cycle of PBMC cells according to the preferred embodiment of the present invention.
FIG. 4 shows the results of the test of the cycle of proliferation of PBMC cells using the four-cycle kit (CN 114487394A) according to the preferred embodiment of the invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
PBMC cells used in the following examples were taken from healthy humans. Flow cytometry was purchased from BD FACSAria (BD, 643181). Alexa Fluro 488-Azide (AAT, 1701) was purchased from AAT.
Example 1 cell five cycle assay kit
The present embodiment provides a cell five-cycle assay kit comprising:
(1) Phosphorylated histone H3 detection reagent: p-H3-Alexa Fluro 647.
(2) Alexa Fluro 488-Azide working fluid:
CuSO 4 ：1mM
Azide-Alexa Fluro 488：2μM
sodium ascorbate: 100mM
Tris-HCl：100mM
(3) Nucleic acid fluorescent dye: DAPI, PY.
(4) EdU solution with working concentration of 10-50. Mu.M.
(5) Cell fixative: 4% paraformaldehyde.
(6) Cell rupture of membranes: PBS+0.5% Triton X-100.
(7) Washing liquid: PBS containing 10% FBS.
Wherein the pH of PBS was 7.4.
Example 2 cell cycle detection method
Isolated healthy human PBMC cells were proliferation stimulated with PMA and Ionomycin for 48h before the start of the experiment.
PMA: myristyl Phorbol ethyl ester (Phorbol-12-myristate-13-acetate).
1. EdU cultured PBMC cells; 10mM EdU solution was added to PMPI cell culture medium (Gibco, 21875034) at a volume ratio of 1:400, and cultured for 4 hours according to the cell proliferation rate;
2. after harvesting the cells and washing them thoroughly with PBS, fixing the cells with 4% paraformaldehyde at room temperature for 20 minutes, and washing them with a washing solution (PBS+10% FBS) to remove the fixing solution;
3. after treating cells with a membrane breaker (PBS+0.5% Triton X-100) for 20 minutes at room temperature, washing with a washing solution to remove the membrane breaker;
4. adding Alexa Fluro 488-Azide working solution into the cells after membrane rupture treatment, staining the cells for 30 minutes at room temperature, and washing the cells with a washing solution;
5. adding p-H3-Alexa Fluro 647 with the final concentration of 1 mug/mL, staining cells for 20 minutes at normal temperature, and fully washing with a washing solution to remove redundant antibodies;
6. cells were resuspended in wash solution, stained with 2. Mu.g/mL DAPI, 0.4. Mu.g/mL PY at room temperature for 10 minutes, and washed well. Cells were resuspended in PBS and prepared for detection on-machine (flow cytometer).
The test results in FIG. 1 show that the PBMC cells can be very clearly separated into five cell cycles G0, G1, S, G and M after the dyeing treatment, and the cells in each cycle have no cross contamination.
Comparative example 1:
1. compared with the conventional three-period kit marked by the first-type nucleic acid reagent in the market
The cell cycle diagram obtained by labeling the conventional first type of nucleic acid reagent is shown in FIG. 2.S and M phase cells are almost difficult to observe, and G1 and G2 cell analysis is unreliable. For a method of labeling a first type of nucleic acid reagent, see Assaying cell cycle status using flow cytomet, kang Ho Kim et al Curr Protoc Mol Biol,2015,1 (11).
2. Compared with the common three-period kit marked by the second nucleotide analogue in the market
The second category labels newly synthesized DNA with nucleotide analogs (EdU, 5-ethynyl-2' deoxyuridine) to distinguish the total DAPI labeled DNA amounts is shown in fig. 3. Although S phase cells can be analyzed, G2/M phase cells cannot be distinguished, and the commercially available kit ThermoFisher Scientific (C10337) is very expensive. For a second method of labelling synthetic DNA with nucleotide analogues, see A chemical method for fast and sensitive detection of DNA synthesis in vivo.
Meanwhile, the existing three-period kit does not relate to the detection of G0-phase cells.
Comparative example 2:
compared with the self-grinding four-cycle kit (CN 114487394 a): the original four-period self-grinding kit adopts a BrdU (5-bromodeoxyuridine) doping labeling method as shown in figure 4, and the required detection time is long and the G0 phase can not be detected by using antibodies corresponding to BrdU.
The novel five-cycle detection kit replaces the BrdU doping labeling method with a second-class nucleic acid analogue (EdU) doping method, and does not need to use BrdU antibodies, so that the detection is simple and quick. Meanwhile, iterative upgrading is carried out on the basis of original four-period detection, G0 phase comparison detection is newly added, comprehensive five-period detection of cells is realized, and the detection result is detailed and reliable.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (9)
1. The five-cycle cell detection kit is characterized by comprising a phosphorylated histone H3 detection reagent, a nucleic acid fluorescent dye, an EdU solution and Alexa Fluro 488-Azide working solution.
2. Kit according to claim 1, wherein the phosphorylated histone H3 detection agent is an anti-phosphorylated histone H3 antibody, preferably p-H3-Alexa fluor 647.
3. The kit of claim 1, wherein the nucleic acid fluorescent dye is selected from DAPI, PY.
4. The kit according to claim 1, wherein the Alexa Fluro 488-Azide working solution contains 1mM CuSO 4 2. Mu.M of Azide-Alexa Fluro 488 and 100mM sodium ascorbate in 100mM Tris.
5. The kit of any one of claims 1-4, further comprising a cell fixative and a cell breaker;
the cell fixative is 2% -4% paraformaldehyde;
the cell rupture agent is PBS containing 0.1-0.5% Triton X-100.
6. Use of the kit of any one of claims 1-5 for cell cycle detection, said use being for non-diagnostic purposes.
7. A method for detecting a cell cycle of non-diagnostic interest, the method comprising:
(1) Inoculating cells into a cell culture medium containing Edu for culture;
(2) Harvesting the cells, washing, and fixing the cells with a cell fixative;
(3) The fixed cells are treated with a cell rupture agent;
(4) Mixing the cells subjected to membrane rupture treatment with Alexa Fluro 488-Azide working solution for reaction;
(5) Washing the harvested cells and incubating with a phosphorylated histone H3 assay reagent;
(6) Washing and re-suspending the cells, adding nucleic acid fluorescent dye for dyeing, washing the cells after dyeing, and carrying out flow cell detection after re-suspending the cells;
wherein the cell fixative, the cell rupture reagent, the phosphorylated histone H3 detection reagent, the Alexa fluor 488-Azide working fluid and the nucleic acid fluorescent dye are derived from the kit of any one of claims 1-5.
8. The method according to claim 7, comprising the steps of:
1) Inoculating the cells into a cell culture medium with the EdU concentration of 10-50 mu M for culturing for 0.5-4 hours;
2) Harvesting cells, washing with PBS, fixing the cells for 10-30 minutes at room temperature with 2% -4% paraformaldehyde, and washing with a washing solution to remove the paraformaldehyde;
3) Treating the cells after fixation with a cell membrane breaker at room temperature for 10-30 minutes, and then washing with a washing solution to remove the membrane breaker;
4) Adding Alexa Fluro 488-Azide working solution into the cells after membrane rupture treatment, staining the cells for 20-30 minutes at room temperature, and washing the cells with a washing solution;
5) Re-suspending the cells in a washing solution, mixing the cells with a phosphorylated histone H3 detection reagent, incubating for 20-30 minutes at normal temperature, and washing the cells with the washing solution;
6) Resuspending the cells in a washing solution, sequentially adding DAPI and PY to make the final concentration of the cells be 0.1-2 mug/mL and 0.1-1 mug/mL respectively, staining the cells for 10-20 minutes at room temperature, washing the cells, resuspending the cells in PBS, and carrying out flow cytometry detection;
the washing solution used in steps 2) to 5) was PBS containing 10% FCS or FBS.
9. The method of claim 7 or 8, wherein the cell is a mammalian somatic cell.
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