Liquid-phase DNA FISH analysis method
By fixing cells with formaldehyde solution and combining 2× SSC buffer and gradient ethanol treatment, the problem of cell rupture in DNA FISH analysis was solved, and the integrity of cell morphology and liquid phase status were achieved, which was suitable for single-cell and gene sequencing analysis. .
Patent Information
- Application Number
- CN202310812579.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-04
- Publication Date
- 2025-01-07
AI Technical Summary
Existing DNA FISH analysis methods can easily lead to cell rupture during cell fixation and staining, especially during high-temperature denaturation, which affects the integrity and accuracy of subsequent single-cell whole genome analysis.
The target cells were fixed for a short time with a formaldehyde solution of 1% to 100%, and 2× SSC buffer was used to replace pure water during high-temperature denaturation, combined with gradient ethanol dehydration treatment to avoid cell rupture and hybridize in situ 2× SSC buffer was used during the procedure to maintain the liquid state of the cells.
It effectively prevents the rupture of cells during high-temperature denaturation and staining, maintains the morphological integrity of the cells, simplifies operations, reduces environmental pollution, and makes the analyzed cells suitable for single-cell extraction and gene sequencing.
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Figure CN119265284A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the fields of biomedical technology and digital medicine, and in particular to a liquid phase DNA FISH (liquid phase nucleic acid in situ hybridization) analysis method, which is used to prevent cell rupture during liquid phase DNA FISH analysis. Background Art
[0002] DNA FISH analysis is commonly used in scientific research and clinical practice. It is an effective means of analyzing the cytological and molecular biological characteristics of isolated target cells. Conventional DNA FISH usually involves centrifuging target cells onto a slide, drying, staining, and analyzing them. If further sequencing or whole-genome analysis is performed on single cells, it will be difficult to obtain complete single cells, and the single-cell whole-genome analysis cannot be performed perfectly. Therefore, how to keep the biological morphology of cells intact to the greatest extent is a key step in obtaining complete single cells after DNA FISH analysis. At present, the reagents commonly used on the market for maintaining cell integrity in DNA FISH are mainly methanol-acetic acid solutions. Alcohols generally cause cells to shrink and deform, which will affect subsequent cell research. Glacial acetic acid, due to its corrosiveness, volatility and pungent smell, often pollutes the experimental environment, causing serious discomfort to the operator's eyes and nasopharynx. Therefore, it is not suitable for long-term use. In addition, methanol-acetic acid solution is difficult to preserve due to its high volatility. It needs to be prepared and used immediately, which is troublesome to operate. Also, due to its high volatility, cells need to be immersed in it to ensure that the cells are fixed in place, and a large amount is often used, which increases the difficulty of handling waste organic solvents after the experiment. Summary of the invention
[0003] In view of the need for cell integrity during liquid-phase DNA FISH analysis, the main purpose of the present invention is to provide a liquid-phase DNA FISH analysis method, which can ensure that the target cells do not break during the high-temperature DNA denaturation and staining operations, which is conducive to the simultaneous analysis of cells at the cell level and in situ gene hybridization. Moreover, the analyzed cells can be used for further analysis such as single cell extraction and whole genome sequencing due to their intact morphological structure.
[0004] In order to achieve the above object, in a basic embodiment, the present invention provides a DNA FISH analysis method in a liquid phase to prevent cell rupture, comprising the following steps:
[0005] S1. Fix the target cells to be subjected to liquid phase DNA FISH analysis in the cell culture dish with cell fixative for 10 to 15 minutes.
[0006] The fixing solution is a formaldehyde solution with a concentration of 1% to 100%, and the amount of the fixing solution is 50-500ul.
[0007] S2. Use the fixed target cells mentioned above to perform immunofluorescence staining and identification of target cells at the protein level.
[0008] S3. Add 2×SSC buffer and soak at room temperature for 10 to 15 minutes.
[0009] S4. Aspirate and discard the 2×SSC buffer solution, and dehydrate by soaking in 70% to 100% ethanol in a gradient concentration for 1 to 2 minutes to replace the water in the cells, making it easier for the probe reagent to immerse into the specimen.
[0010] Dehydration using gradient ethanol concentrations is more gentle. Direct use of high-concentration alcohol will cause the cells to dehydrate and shrink rapidly and become hard and brittle, which is not conducive to maintaining the integrity of the cell structure.
[0011] S5. Aspirate and discard the ethanol solution, rinse once with 2×SSC buffer, and immediately add the in situ hybridization probe reagent. Be careful to avoid light, and add pure water in the gaps between the wells to maintain the humidity in the wells to prevent the reagent from volatilizing.
[0012] S6. Place the cell culture plate into a ziplock bag, immerse it in a water bath at 82-83°C for 10-12 min, and then transfer it to a water bath at 50-52°C for 1.5-2 h.
[0013] S7. Wash once with 2×SSC buffer, add DAPI staining solution to the cell culture plate for counterstaining, let stand for 3-5 minutes, and add 2×SSC buffer.
[0014] S8. Immediately place the cell culture plate under a fluorescence microscope for imaging observation and image taking.
[0015] Optionally, the target cells to be subjected to DNA FISH liquid phase analysis include cultured cell line cells such as MCF-7, MBA-MD-231 breast cancer cells, HepG2 liver cancer cells, A549 lung cancer cells, etc., enriched and separated circulating tumor cells (CTCs) or single cells of tissue origin.
[0016] Optionally, the immunofluorescence staining identification of the target cell protein level can use an analysis kit product for marker labeling and staining of cell membrane proteins.
[0017] The 2×SSC buffer described in step S3 of the present invention is superior to pure water or ddH2O and can stabilize cells in a liquid phase without rupture.
[0018] Through the above technical scheme, the present invention can completely avoid the loss of research objects due to cell rupture during high temperature denaturation and staining when performing liquid phase DNA FISH analysis on target cells, especially when there are only a few or even one target cells in the patient sample, accurate analysis of cells is particularly important. The present invention can achieve the purpose of cell fixation by adding a trace amount of 4% formaldehyde solution to the separated target cells for a short time treatment, the operation is simple, and the cells do not rupture at all during the subsequent high temperature denaturation process, and the cell morphology integrity rate is high. During the staining operation, 2×SSC buffer is used instead of ddH2O to keep the cells in a liquid phase, which effectively reduces cell rupture and has strong practicality in clinical and scientific research.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] The method of the present invention uses a trace amount of 4% formaldehyde solution to fix the target cells for a short time, so that proteins and other substances in the specimen are quickly denatured. In the later processing, the morphology and properties will not be changed, and the cell rupture during DNA FISH liquid phase analysis is prevented, and the morphological integrity of the cells in the high-temperature DNA denaturation process is fully guaranteed. The operation is simple, environmental pollution will not be caused, and a large amount of organic waste liquid will not be generated. In addition, in order to ensure that the cells are always in the liquid phase condition to maintain the original three-dimensional morphology, after the gradient ethanol treatment, in order to avoid the rapid volatilization of ethanol causing the cells to shrink and stick to the bottom plate, it is necessary to immediately add 2×SSC buffer to restore the cell liquid phase environment. The salt ions (Na) in the SSC buffer neutralize the negative charges on the RNA / DNA main chain, which can improve the binding efficiency of the probe and the target sequence. It is often used in the hybridization process to achieve the purpose of denaturation and washing. Therefore, it is more appropriate to select 2×SSC buffer for cell preservation. The cells that have been fixed and identified through liquid phase staining fully maintain their original three-dimensional morphology and are in a suspended state, which is conducive to accurate analysis of the cells at both the cellular and genetic levels. Moreover, since the analyzed cells have intact morphological structure, they can also be used for single-cell extraction and gene sequencing analysis. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 Schematic diagram comparing the cell states of the fixed MCF-7 cells before and after high temperature denaturation in Example 1 of the present invention, wherein (a) is before high temperature denaturation, and (b) is after high temperature denaturation.
[0022] Figure 2 Schematic diagram of the comparison of the cell states of unfixed MCF-7 cells before and after high temperature denaturation in Control Example 1 of the present invention, wherein (a) is before high temperature denaturation, and (b) is after high temperature denaturation.
[0023] Figure 3 This is a schematic diagram of liquid phase immunostaining combined with FISH staining of MCF-7 cells with nanomagnetic beads in a cell culture plate in Example 2 of the present invention.
[0024] Figure 4 This is a schematic diagram of liquid phase immunostaining combined with FISH staining of MCF-7 nude cells in a cell culture plate in Example 2 of the present invention.
[0025] Figure 5 This is a schematic diagram of MCF-7 naked cell immunostaining combined with FISH staining using the traditional glass slide method in comparison example 3 of the present invention. DETAILED DESCRIPTION
[0026] In view of the requirement for cell integrity during DNA FISH liquid phase analysis, the main idea of the embodiments of the present invention is:
[0027] In order to prevent cell rupture during the operation, the present invention provides a liquid phase DNA FISH analysis method, comprising the following steps:
[0028] S1. Fix the target cells to be subjected to liquid DNA FISH analysis in a cell culture dish with 50 to 500 ul of a 1% to 100% formaldehyde solution for 10 to 15 minutes; preferably, a 4% formaldehyde solution.
[0029] S2. Use the fixed target cells mentioned above to perform immunofluorescence staining and identification of target cells at the protein level.
[0030] S3. Add 2×SSC buffer and soak at room temperature for 10 to 15 minutes.
[0031] S4. Aspirate and discard the 2×SSC buffer and soak in 70% to 100% ethanol in gradient concentrations for 1 to 2 minutes.
[0032] S5. Aspirate and discard the ethanol solution, rinse once with 2×SSC buffer, and immediately add the in situ hybridization probe reagent. Be careful to avoid light, and add pure water in the gaps between the wells to maintain the humidity in the wells to prevent the reagent from volatilizing.
[0033] S6. Place the cell culture plate into a ziplock bag, immerse it in a water bath at 82-83°C for 10-12 min, and then transfer it to a water bath at 50-52°C for 1.5-2 h.
[0034] S7. Wash once with 2×SSC buffer, add DAPI staining solution to the cell culture plate for counterstaining, let stand for 3-5 minutes, and add 2×SSC buffer.
[0035] S8. Immediately place the cell culture plate under a fluorescence microscope for imaging observation and image taking.
[0036] Among them, the embodiment of the present invention uses cultured breast cancer cell line MCF-7 cells. The MCF-7 cells obtained by digestion and collection are used to prove the effectiveness of the fixative in maintaining the integrity of cells during high-temperature denaturation, that is, about 100 fixed and fresh tumor cells are respectively aspirated, placed in a cell culture plate, incubated in an 82°C water bath for 10 minutes, and the cell integrity rate is observed and counted under a microscope; on the other hand, it is used to prove the positive role of 2×SSC buffer in maintaining cell stability.
[0037] The present invention will be described in detail below in conjunction with specific examples. The materials used in the examples can be obtained from commercial channels, wherein the blood cell analysis kit and cell sorter were purchased from Yichang Micron Silicon Valley Life Science Co., Ltd.
[0038] Example 1
[0039] This embodiment provides a DNA FISH liquid phase analysis method for preventing cells from rupturing during the high temperature denaturation process of DNA FISH, which specifically includes the following steps:
[0040] S1. Collect the cells obtained by digesting the cultured MCF-7 cells into a 15 ml centrifuge tube.
[0041] S2. Add 100ul of 4% formaldehyde solution to the above 15ml centrifuge tube, shake and mix, let it stand for 10 minutes, and add 1ml of 1XPBS to make a cell suspension.
[0042] S3. Dilute the fixed MCF-7 cell suspension to a concentration of 5 to 10 cells / ul for later use.
[0043] S4. Take about 100 fixed MCF-7 cells and place them in a six-well cell culture plate. Observe and count the cells under a microscope. The results are as follows: Figure 1 The counting results are shown in Table 1.
[0044] S5. Place the above six-well cell culture plate in an 82° C. water bath and incubate for 10 min.
[0045] S6. Observe and count the number of intact cells remaining under a microscope. The results are as follows: Figure 1 The counting results are shown in Table 1.
[0046] Figure 1 The diagram is a comparison diagram of the cell states of the fixed MCF-7 cells before and after high temperature denaturation in Example 1 of the present invention. The left side of the figure shows the fixed MCF-7 cells, and the right side shows the fixed MCF-7 cells after high temperature denaturation. It can be seen that the fixed MCF-7 cells did not break under the high temperature denaturation conditions and still maintained intact cell morphology.
[0047] Table 1 shows the intact rate of the fixed MCF-7 cells after high temperature denaturation in Example 1 of the present invention. The data in the table include the initial cell number, the number of intact cells remaining after high temperature denaturation, the cell intact rate of three repeated tests, and the average intact rate, where the cell intact rate = the number of intact cells remaining after high temperature denaturation / the initial cell number. It can be seen from the table that the cell intact rate of the fixed MCF-7 cells after the high temperature water bath remains 100%.
[0048] Table 1 Intact rate of fixed MCF-7 cells after high temperature denaturation
[0049]
[0050] Control experiment 1
[0051] This experiment follows the method in Example 1, using unfixed cells to perform a high temperature denaturation experiment, specifically including the following steps:
[0052] S1. Collect the cells obtained by digesting the cultured MCF-7 cells into a 15 ml centrifuge tube.
[0053] S2. Add 1 ml of 1XPBS to the above 15 ml centrifuge tube to prepare a cell suspension.
[0054] S3. Dilute the above MCF-7 cell suspension to a concentration of 5-10 cells / ul for later use.
[0055] S4. Take about 100 MCF-7 cells and place them in a six-well cell culture plate. Observe and count the cells under a microscope. The results are as follows: Figure 2 The counting results are shown in Table 2.
[0056] S5. Place the above six-well cell culture plate in an 82° C. water bath and incubate for 10 min.
[0057] S6. Observe and count the number of intact cells remaining under a microscope. The results are as follows: Figure 2 The counting results are shown in Table 2.
[0058] Figure 2 This is a schematic diagram showing the cell status comparison of the unfixed MCF-7 cells before and after high temperature denaturation in the control experiment 1 of the present invention. Figure 2 (a)) shows MCF-7 cells with intact morphology. Figure 2 (b) shows the MCF-7 cells that were broken and deformed after high temperature denaturation. It can be seen that most of the unfixed MCF-7 cells have been broken after high temperature denaturation.
[0059] Table 2 shows the intact rate of unfixed MCF-7 cells after high temperature denaturation in control experiment 1 of the present invention. The data in the table include the initial cell number, the number of intact cells remaining after high temperature disruption, the cell intact rate of three repeated experiments, and the average intact rate, where the cell intact rate = the number of intact cells remaining after high temperature denaturation / the initial cell number. It can be seen from the table that the average intact rate of unfixed MCF-7 cells is only 24%.
[0060] Table 2 Intact rate of unfixed MCF-7 cells after high temperature denaturation
[0061]
[0062]
[0063] Example 2
[0064] This embodiment provides a DNA FISH liquid phase analysis method for preventing cell rupture during DNA FISH liquid phase analysis, which specifically includes the following steps:
[0065] S1. Take fresh MCF-7 cell suspension, add EPCAM nanomagnetic beads and incubate to make the cell surface adhere to specific antibody magnetic beads, count the cell concentration, take about 100 tumor cells adhered to magnetic beads and place them in the three wells on the left side of the six-well cell culture plate, and take about 100 MCF-7 naked cells and place them in the three wells on the right side, and count the number of cells in each well under a microscope.
[0066] S2. Place the cell culture plate in the magnetic field of the cell sorter, add 100ul of 4% formaldehyde solution to each well for fixation for 10 minutes, and discard the fixative.
[0067] S3. Use a blood cell analysis kit to stain the tumor cells in each well with tumor antigen markers, and discard the staining solution in a magnetic field.
[0068] S4. Take 2 ml of 2×SSC buffer and slowly add it into the well from the edge of the well plate. Leave it at room temperature for 10 minutes and discard the buffer in a magnetic field.
[0069] S5. Add 70% ethanol, 90% ethanol and 100% ethanol respectively and soak for 2 min in sequence, then discard the ethanol in a magnetic field.
[0070] S6. Add 1 ml of 2×SSC buffer to rinse once, use a pipette to take 10 ul of chromosome 7 probe reagent and drop it in the center of each well, shake it slightly to allow the solution to cover the target area, pay attention to avoid light, and add pure water in the gaps between the wells to maintain the humidity in the wells to prevent the reagent from volatilizing.
[0071] S7. Place the cell culture plate into an aluminum foil bag, immerse it in an 82°C water bath and incubate for 10 min, then transfer it to a 50°C water bath and immerse it in incubation for 1.5 h.
[0072] S8. Wash once with 2×SSC buffer, add DAPI staining solution to the cell culture plate for counterstaining, let stand for 3 minutes, and slowly add 1 ml of 2×SSC buffer along the wall of the well.
[0073] S9. Immediately place the cell culture plate under a fluorescence microscope and observe the image. Figure 3 , Figure 4 The counting results are shown in Table 3.
[0074] Figure 3 This is a schematic diagram of liquid phase immunostaining combined with FISH staining of MCF-7 cells with nanomagnetic beads in a cell culture plate in Example 2 of the present invention. Figure 3 In the figure, BF indicates the detection result of EpCAM nano-antibody magnetic beads combined with tumor cells under a fluorescence microscope, DAPI indicates the detection result of cell nuclei stained with DAPI under a fluorescence microscope (cell nuclei are blue in the figure), CK indicates the detection result of keratin stained with CK under a fluorescence microscope (cells are green in the figure), CSP7 indicates the detection result of the number of chromosomes 7 under a fluorescence microscope (chromosomes in the cell nuclei are green in the figure), CD45 indicates the result of detecting white blood cells of extracted tumor cells under a fluorescence microscope, and Merge indicates the image obtained by superimposing the four images marked with DAPI, CK, CSP7, and CD45. Figure 3 As shown, after immunofluorescence staining and fluorescence in situ hybridization staining, nanomagnetic beads specifically adhered to the cell surface can be seen under bright field, with blue fluorescence (cell nucleus stained with DAPI), green fluorescence (keratin stained with CK) and green fluorescent dots (greater than 2) or clusters (chromosomes stained with CSP7), and no red fluorescence (CD45 identifies white blood cells), indicating that the detected cells are specifically labeled by EpCAM antibody magnetic beads, have cell nuclei, and are intact cells; and exhibit abnormal polyploidy of chromosome 7, indicating that the detected cells are tumor cells.
[0075] Figure 4 This is a schematic diagram of liquid phase immunostaining combined with FISH staining of MCF-7 nude cells in a cell culture plate in Example 2 of the present invention. Figure 4In the figure, BF indicates the intact cell outline of tumor cells under bright field, DAPI indicates the detection result of cell nuclei stained with DAPI under fluorescence microscope (cell nuclei are blue in the figure), CK indicates the detection result of keratin stained with CK under fluorescence microscope (cells are green in the figure), CSP7 indicates the detection result of chromosome 7 under fluorescence microscope (chromosomes in cell nuclei are green in the figure), CD45 indicates the result of detecting white blood cells of extracted tumor cells under fluorescence microscope, and Merge indicates the image obtained by superimposing four images marked with DAPI, CK, CSP7, and CD45. Figure 4 As shown, after immunofluorescence staining and fluorescent in situ hybridization staining, the cell outline is clear and intact under the bright field, with blue fluorescence (cell nucleus stained by DAPI), green fluorescence (keratin stained by CK) and green fluorescent dots (greater than 2) or clusters (chromosomes stained by CSP7), and no red fluorescence (CD45 identifies white blood cells), indicating that the detected cells have intact morphology, cell nuclei, and are intact cells; and they show abnormal polyploidy of chromosome 7, indicating that the detected cells are tumor cells.
[0076] Table 3 shows the tumor cell detection rate of MCF-7 cells with nanometer magnetic beads and MCF-7 cells in Example 2 of the present invention after liquid phase immunostaining and DNA FISH in cell culture plates, i.e., the DNA FISH detection rate when wetting or washing with 2×SSC buffer. The data in the table include the initial cell number, the number of cells detected after cell and chromosome staining, the cell detection rate of repeated tests three times, and the average detection rate, and the tumor cell detection rate = the number of tumor cells detected after cell and chromosome staining / the initial number of tumor cells. It can be seen from the table that the detection rate of tumor cells adhered to magnetic beads stably in the magnetic field for cell and chromosome staining reached 91%, while the cell loss rate of cells adhered to the surface of the cell culture plate by the cells themselves during DNA FISH staining reached 11%.
[0077] Table 3 DNA FISH detection rate when using 2×SSC solution for wetting or washing
[0078]
[0079]
[0080] Control experiment 2
[0081] This experiment follows the method in Example 2, using ddH2O for cell wetting and washing, specifically including the following steps:
[0082] S1. Take fresh MCF-7 cell suspension, count the cell concentration, take about 100 tumor cells and place them in a six-well cell culture plate, and count the number of cells in each well under a microscope.
[0083] S2. Add 100ul of 4% formaldehyde solution to each well of the above cell culture plate and fix for 10 minutes, then discard the fixative.
[0084] S3. Use a blood cell analysis kit to stain the tumor antigen markers of each well of tumor cells, and discard the staining solution.
[0085] S4. Take 2 ml of 2×SSC buffer and slowly add it into the wells from the edge of the well plate. Leave it at room temperature for 10 minutes and then aspirate and discard the solution.
[0086] S5. Add ddH2O, 70% ethanol, 90% ethanol and 100% ethanol respectively, soak for 2 min in sequence and then discard.
[0087] S6. Use a pipette to take 10ul of chromosome 7 probe reagent and drop it in the center of each well. Shake slightly to allow the solution to cover the target area. Be careful to avoid light, and add pure water to the gaps between the wells to maintain the humidity in the wells to prevent the reagent from volatilizing.
[0088] S7. Place the cell culture plate into an aluminum foil bag, immerse it in an 82°C water bath and incubate for 10 min, then transfer it to a 50°C water bath and immerse it in incubation for 1.5 h.
[0089] S8. Wash once with ddH2O, add DAPI staining solution to the cell culture plate for counterstaining, let stand for 3 minutes, and slowly add 1 ml of 2×SSC buffer along the wall of the well.
[0090] S9. Immediately place the cell culture plate under a fluorescence microscope for observation. The counting results are shown in Table 3.
[0091] Table 4 shows the DNA FISH detection rate when ddH2O is used for wetting or washing in control experiment 2 of the present invention. The data in the table include the initial cell number, the number of cells detected after cell and chromosome staining, the cell detection rate of three repeated tests, and the average detection rate. The tumor cell detection rate = the number of tumor cells detected after cell and chromosome staining / the initial tumor cell number. It can be seen from the table that the cell loss rate of tumor cells and naked cells attached to magnetic beads during DNA FISH staining exceeded 10%, indicating that ddH2O is not suitable for cell washing and preservation.
[0092] Table 4 DNA FISH detection rate when wetting or washing with ddH2O
[0093]
[0094]
[0095] Control experiment 3
[0096] This experiment followed the method of control experiment 2, centrifuging cells onto adhesion slides for analysis, including the following steps:
[0097] S1. Take fresh MCF-7 cells, add 100ul 4% formaldehyde solution to treat for 10 minutes, add 1ml 1×PBS solution to make a suspension, take about 100 tumor cells and add them to 1ml 1×PBS to make a sample to be tested.
[0098] S2. Place the sample to be tested into a centrifugal funnel, assemble the funnel and the attached glass slide, and then place the assembled funnel into a glass slide centrifuge and centrifuge at 1500 rpm for 10 min.
[0099] S3. Take out the slide, count the number of cells on the slide under a microscope, and then let it dry naturally.
[0100] S4. Use a blood cell analysis kit to stain the tumor cells on the slide for tumor antigen markers.
[0101] S5. Soak the slides in ddH2O, 70% ethanol, 90% ethanol, and 100% ethanol for 2 min respectively and then dry them.
[0102] S6. Use a pipette to take 10ul of the probe reagent and drop it on the slide. Cover it with a coverslip and avoid light.
[0103] S7. Place the slide in a wet box, put the wet box into a ziplock bag, immerse it in an 82°C water bath and incubate for 10 min, then transfer it to a 50°C water bath and immerse it in incubation for 1.5 h.
[0104] S8. Take out the slide, soak it in ddH2O to remove the cover glass, add 100ul DAPI to cover the target area, place it at room temperature away from light for 3 minutes, aspirate and discard the staining solution, add the sealing agent, and cover with a cover glass.
[0105] S9. Immediately place the slide under a fluorescence microscope for observation and image taking.
[0106] Figure 5 This is a schematic diagram of liquid phase immunostaining combined with FISH staining of MCF-7 nude cells adhered to a glass slide in control experiment 3 of the present invention. Figure 5In the figure, BF indicates the intact cell outline of tumor cells under bright field, DAPI indicates the detection result of cell nuclei stained with DAPI under fluorescence microscope (cell nuclei are blue in the figure), CK indicates the detection result of keratin stained with CK under fluorescence microscope (cells are green in the figure), CSP7 indicates the detection result of chromosome 7 under fluorescence microscope (chromosomes in cell nuclei are green in the figure), CD45 indicates the result of detecting white blood cells of extracted tumor cells under fluorescence microscope, and Merge indicates the image obtained by superimposing four images marked with DAPI, CK, CSP7, and CD45. Figure 5 As shown, after immunofluorescence staining and fluorescent in situ hybridization staining, the cell outline is clear and intact under the bright field, with blue fluorescence (cell nucleus stained by DAPI), green fluorescence (keratin stained by CK) and green fluorescent dots (greater than 2) or clusters (chromosomes stained by CSP7), and no red fluorescence (CD45 identifies white blood cells), indicating that the detected cells have intact morphology, cell nuclei, and are intact cells; and they show abnormal polyploidy of chromosome 7, indicating that the detected cells are tumor cells.
[0107] Table 5 shows the DNA FISH detection rate of the conventional glass slide method in control experiment 3 of the present invention. The data in the table include the initial cell number, the number of cells detected after cell and chromosome staining, the cell detection rate of three repeated tests, and the average detection rate. The tumor cell detection rate = the number of tumor cells detected after cell and chromosome staining / the initial tumor cell number. It can be seen from the table that the detection rate of DNA FISH staining after centrifuging naked cells onto an adhesive glass slide and drying is higher than that in a cell culture plate, indicating that the adhesive surface can effectively increase the adhesion of naked cells on the surface of consumables, but the glass slide method requires staining operation after drying, and a cover glass needs to be added after staining, which is not conducive to subsequent single cell extraction and gene analysis. The liquid phase DNA FISH method in the cell culture plate just overcomes this defect, and the cells after magnetic bead labeling can complete the analysis of cell level and chromosome level in the liquid phase environment throughout the process, and the cell integrity rate is higher than the staining method on the glass slide, so the present invention can perform the greatest depth analysis of rare cell samples, filling the gap in domestic related technologies.
[0108] Table 5 DNA FISH detection rate of the traditional slide method
[0109]
Claims
1. A liquid phase DNA FISH analysis method, characterized in that: The following steps are involved: S1. Fix the target cells in the cell culture dish to be subjected to liquid phase DNA FISH analysis with a cell fixative for 10 to 15 minutes; the fixative is a formaldehyde solution with a concentration of 1% to 100%, and the amount used is 50-500ul; S2. Using the fixed target cells mentioned above to perform immunofluorescence staining and identification of target cells at the protein level; S3, add 2×SSC buffer and soak at room temperature for 10-15 minutes; S4, discard the buffer solution, and soak in 70% to 100% ethanol in gradient concentrations for 1 to 2 minutes; S5. Aspirate and discard the ethanol solution, rinse once with 2×SSC buffer, add in situ hybridization probe reagent, avoid light, and add pure water in the gaps between the wells to maintain the humidity in the wells to prevent the reagent from volatilizing; S6. Place the cell culture plate in a ziplock bag, immerse it in a water bath at 82-83°C for 10-12 min, and then transfer it to a water bath at 50-52°C for 1.5-2 h. S7, washing once with 2×SSC buffer, adding DAPI staining solution to the cell culture plate for counterstaining, standing for 3-5 minutes, and adding 2×SSC buffer; S8. Immediately place the cell culture plate under a fluorescence microscope for imaging observation and image taking.
2. The method according to claim 1, characterized in that In step S1, the target cells to be subjected to liquid phase DNA FISH analysis include cultured cell line cells, circulating tumor cells (CTCs) obtained by enrichment and separation, or single cells derived from tissues.
3. The method according to claim 2, characterized in that The cultured cell lines include MCF-7, MBA-MD-231 breast cancer cells, HepG2 liver cancer cells, and A549 lung cancer cells.
4. The method according to claim 1, characterized in that: In step S2, the immunofluorescence staining identification of the target cell protein level uses an analysis kit product for marker labeling and staining of cell membrane proteins.