Kit for preparing circulating tumor cell sorting quality control product, preparation method and cell sorting method
By using cell labeling reagents and buffers containing DMSO solvents, lipophilic dyes and staining enhancers, cell preservation agents with glucose, the problems of low efficiency and waste of antibodies in the preparation of circulating tumor cell sorting quality control products are solved, and high efficiency is achieved The cell marking and preservation of cells prolongs cell activity and improves sorting efficiency.
Patent Information
- Application Number
- CN202411292071.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-14
- Publication Date
- 2025-01-07
AI Technical Summary
The existing preparation methods for circulating tumor cell sorting quality control products are inefficient and antibodies are wasted, and the cell storage temperature is too low to affect cell activity, resulting in low sorting efficiency.
A kit is provided, including cell labeling reagents and cell preservation agents, which consist of DMSO solvents, lipophilic dyes and staining enhancers, and cell preservation agents consist of buffer solution and glucose for labeling and preserving positive quality-controlled cells. , ensure that the cells are stored at 0-4°C for more than one month, and avoid subsequent antibody staining and identification.
It improves the efficiency of circulating tumor cell sorting, reduces the waste of antibodies, and maintains cell activity through appropriate storage conditions, extending the service life of the quality control product.
Smart Images
Figure CN119265269A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of biological preparations, and in particular to a kit for preparing a circulating tumor cell sorting quality control product, a preparation method, and a cell sorting method. Background Art
[0002] As a liquid biopsy method, the detection of circulating tumor cells (CTC) has a wide range of applications in early screening, diagnosis, and prognosis of tumors. The quality control products of the stable detection system provide a good reference and evaluation system for the sorting system, providing a guarantee for the next step of sample testing.
[0003] Most of the quality control products currently used are simulated CTC detection and sorting after cell preservation, and downstream antibody immunofluorescence staining is performed to distinguish the cell sorting effect, which is not conducive to the efficiency of sorting and also wastes the subsequent antibody identification. In addition, most of the current quality control cells are stored at a temperature below -20 degrees, which is not conducive to the preservation of cell activity and affects the sorting efficiency. Summary of the invention
[0004] The main purpose of the present application is to provide a kit, a preparation method, and a cell sorting method for preparing circulating tumor cell sorting quality control products, aiming to solve the technical problems of low sorting efficiency and antibody waste in existing cell sorting methods.
[0005] To achieve the above objectives, the present application provides a kit for preparing a circulating tumor cell sorting quality control product, comprising: independently existing cell labeling reagents and cell preservatives, wherein the cell labeling reagents include DMSO solvent, lipophilic dye and staining enhancer, and the cell preservatives include buffer and glucose.
[0006] In one embodiment, the lipophilic dye includes lipophilic carbocyanine dye, lipophilic porphyrin compound, lipophilic polycyclic aromatic hydrocarbon, lipophilic phthalocyanine compound.
[0007] In one embodiment, the staining enhancer comprises a surfactant and a pegylation agent.
[0008] In one embodiment, the buffer comprises citrate buffer, phosphate buffer, HEPES buffer, Tris buffer or MOPS buffer.
[0009] In one embodiment, the buffer is a citrate buffer, and the ratio between citric acid and sodium citrate in the buffer is 1:1-1:3.
[0010] In addition, to achieve the above-mentioned purpose, the present application also provides a method for preparing a circulating tumor cell sorting quality control product, which is prepared using the kit for preparing a circulating tumor cell sorting quality control product as described above, and the method comprises:
[0011] The positive quality control cells are stained with a cell labeling reagent, and the stained positive quality control cells are stored in a cell preservative;
[0012] Take negative quality control cells and store them in cell preservative;
[0013] The stored negative quality control cells and positive quality control cells were mixed to obtain circulating tumor cell sorting quality control products.
[0014] In one embodiment, the buffer solution accounts for 55% and the glucose accounts for 45% in the cell preservative.
[0015] In one embodiment, the step of staining positive quality control cells using the cell labeling reagent comprises:
[0016] The digested positive quality control cells were collected, incubated with cell labeling reagent for 30 min, and then washed three times with PBS.
[0017] In one embodiment, the stained positive quality control cells are stored in the cell preservative at a temperature of 0-4° C., and the storage time for maintaining cell activity is at least 1 month.
[0018] In addition, to achieve the above objectives, the present application also provides a cell sorting method, the method comprising:
[0019] The quality control cells prepared as described above were incubated with the immunomagnetic beads at 4°C for 2 h;
[0020] According to the CTC sorting procedure, the incubated magnetic beads and quality control cells are taken for sorting on the machine. After the sorting is completed, the obtained magnetic beads are collected and slices are made. They are viewed and counted under immunofluorescence to obtain the cell sorting rate.
[0021] The kit for preparing circulating tumor cell sorting quality control products provided by the present application includes an independently existing cell labeling reagent and a cell preservative, wherein the cell labeling reagent includes a DMSO solvent, a lipophilic dye and a staining enhancer, and the cell preservative includes a buffer and glucose. The cell labeling reagent is used to mark positive cells in the process of preparing quality control products, wherein the DMSO solvent is used to dissolve the lipophilic dye and the staining enhancer, the lipophilic dye is used to stain the cells, and after entering the cell membrane, it can diffuse laterally to gradually stain the cell membrane of the entire cell, and it is very stable after staining, and the staining enhancer accelerates the cell staining speed, making the fluorescent staining brighter and the staining background lower, and the cell preservative is used to preserve negative cells and positive cells after staining, and obtain circulating tumor cell sorting quality control products, the quality control product cells are preserved in the cell preservative, the buffer is used to adjust and stabilize the acid-base environment of the solution, and glucose provides energy for cell survival, and the positive cells in the quality control product are preserved after staining, and the countable cells of the labeled positive cells can be directly provided in the process of cell sorting, and the sorting rate can be directly calculated to avoid the next step of antibody staining identification, so the sorting efficiency is improved, and the waste of antibodies is also avoided. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present drawings or related technologies, the drawings required for use in the embodiments or related technical descriptions will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present drawings. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.
[0023] Figure 1 This is a fluorescence image of cell nuclei after staining with a cell labeling reagent prepared with 1 μM DIO;
[0024] Figure 2 The fluorescence image of the cell labeling reagent prepared with 1 μM DIO;
[0025] Figure 3 This is a picture of cell morphology after staining with a cell labeling reagent prepared with 1 μM DIO;
[0026] Figure 4 This is a fluorescence image of cell nuclei after staining with a cell labeling reagent prepared with 10 μM DIO;
[0027] Figure 5 Fluorescence image of cell labeling reagent prepared with 10 μM DIO;
[0028] Figure 6 This is a picture of cell morphology after staining with a cell labeling reagent prepared with 10 μM DIO;
[0029] Figure 7This is a fluorescence image of cell nuclei after staining with a cell labeling reagent prepared with 30 μM DIO;
[0030] Figure 8 Fluorescence image of cell labeling reagent prepared with 30 μM DIO;
[0031] Fig. 9 This is a picture of cell morphology after staining with a cell labeling reagent prepared with 30 μM DIO;
[0032] Fig.10 This is a fluorescence image of the cell nucleus of A431 cells stored in a cell preservative at 4°C for 7 days;
[0033] Fig.11 This is a fluorescence image of cell labeling reagents for A431 cells stored in a cell preservative at 4°C for 7 days;
[0034] Fig.12 The cell morphology of A431 cells stored in a cell preservative at 4°C for 7 days;
[0035] Fig.13 This is a fluorescence image of the cell nucleus of A431 cells stored in a cell preservative at 4°C for 15 days;
[0036] Fig.14 This is a fluorescence image of cell labeling reagents for A431 cells stored in a cell preservative at 4°C for 15 days;
[0037] Fig.15 The cell morphology of A431 cells stored in cell preservative at 4°C for 15 days;
[0038] Fig.16 This is a fluorescence image of the cell nucleus of A431 cells stored in a cell preservative at 4°C for 30 days;
[0039] Fig.17 This is a fluorescence image of cell labeling reagents for A431 cells stored in a cell preservative at 4°C for 30 days;
[0040] Fig.18 The cell morphology of A431 cells stored in cell preservative at 4°C for 30 days;
[0041] Fig.19 This is a cell morphology picture of ball-1 cells stored in a cell preservative at 4°C for 7 days;
[0042] Fig. 20 This is a fluorescence image of the cell nucleus of ball-1 cells stored in a cell preservative at 4°C for 7 days;
[0043] Fig.21This is a cell morphology picture of ball-1 cells stored in a cell preservative at 4°C for 15 days;
[0044] Fig. 22 This is a fluorescence image of the cell nucleus of ball-1 cells stored in a cell preservative at 4°C for 15 days;
[0045] Fig.23 This is a cell morphology picture of ball-1 cells stored in a cell preservative at 4°C for 30 days;
[0046] Fig.24 This is a fluorescence image of cell nuclei of ball-1 cells stored in cell preservative at 4°C for 30 days;
[0047] Fig.25 This is the fluorescence image of the cell nuclei of the quality control product for cell sorting on the 7th day of storage;
[0048] Fig.26 This is a fluorescence image of the cell labeling reagent for cell sorting on the 7th day of storage;
[0049] Fig. 27 This is the fluorescence image of the cell nuclei of the quality control product for cell sorting on the 15th day of storage;
[0050] Fig.28 This is a fluorescence image of the cell labeling reagent used for cell sorting on the 15th day of storage;
[0051] Fig.29 This is the nuclear fluorescence image of the quality control product for cell sorting on the 30th day of storage;
[0052] Fig.30 This is a fluorescence image of cell labeling reagents used for cell sorting on the 30th day of storage.
[0053] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION
[0054] It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.
[0055] Below, the kit for preparing a circulating tumor cell sorting quality control product, the preparation method, and the cell sorting method of the present application are specifically disclosed in detail with appropriate reference to the accompanying drawings. However, there may be cases where unnecessary detailed descriptions are omitted. For example, there are cases where detailed descriptions of well-known matters and repeated descriptions of actually the same structure are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the drawings and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter described in the claims.
[0056] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and understandable, the technical solution of the present application is further described below in conjunction with the accompanying drawings and embodiments. However, the present application is not limited to the listed embodiments, and should also include any other known changes within the scope of the rights claimed in the present application.
[0057] An embodiment of the present application provides a kit for preparing a circulating tumor cell sorting quality control product, comprising: an independently existing cell labeling reagent and a cell preservative, wherein the cell labeling reagent comprises a DMSO solvent, a lipophilic dye and a staining enhancer, and the cell preservative comprises a buffer and glucose.
[0058] Cell labeling reagent is used to mark positive cells in the process of preparing quality control products, which is convenient for subsequent cell sorting. The cell labeling reagent of the present embodiment includes DMSO solvent, lipophilic dye and staining enhancer. DMSO solvent is used to dissolve lipophilic dye and staining enhancer. The main component of cell membrane is phospholipid, and the dye molecule in lipophilic dye can dye cell membrane, and after entering the cell membrane, it can diffuse laterally and gradually make the cell membrane of the whole cell be dyed, which is very stable after dyeing. Staining enhancer can make cell membrane staining faster, fluorescent staining brighter, and staining background lower. The combination of lipophilic dye and staining enhancer can make cell staining effect more firm and staining time lasting.
[0059] Optionally, the lipophilic dye includes lipophilic carbocyanine dye, lipophilic porphyrin compound, lipophilic polycyclic aromatic hydrocarbon, lipophilic phthalocyanine compound. Lipophilic carbocyanine dye has a long lipophilic hydrocarbon chain and good dyeing effect. Different substances can be selected according to the dyeing characteristics of these lipophilic dyes, and the activity of the stained cells can be avoided to be affected.
[0060] Optionally, the staining enhancer includes a surfactant and a pegylated agent. The surfactant helps the dye to better penetrate and distribute on the cell membrane, such as Triton X-100, which can increase the cell membrane's uptake of the dye and improve the brightness and uniformity of the staining. The pegylated agent can increase the fluorescence intensity and stability of the cell membrane, thereby enhancing the staining effect.
[0061] In this embodiment, the cell preservative includes a buffer and glucose. The buffer is used to adjust and stabilize the pH value of the solution, and glucose is the main energy source for cells, ensuring the activity and function of cells during the preservation period. At the same time, the appropriate glucose concentration can also help maintain the osmotic pressure of the cells and prevent changes in cell activity or morphology.
[0062] Optionally, the buffer used includes citrate buffer, phosphate saline (PBS) buffer, HEPES (4-hydroxyethylpiperazineethanesulfonic acid) buffer, Tris (tris(hydroxymethylaminomethane)) buffer or MOPS (3-morpholinepropanesulfonic acid) buffer.
[0063] Optionally, the buffer used is a citrate buffer, wherein the ratio between citric acid and sodium citrate in the buffer is 1:1-1:3. A 1:1 ratio is more commonly used and can provide a certain range of pH buffering capacity in some cases. A 1:3 ratio may also be used in specific formulations to achieve a specific acidity or buffering effect.
[0064] In the kit of this embodiment, the cell labeling reagent is used to label positive cells in the process of preparing quality control products, wherein the DMSO solvent is used to dissolve the lipophilic dye and the staining enhancer, the lipophilic dye is used to stain the cells, and after entering the cell membrane, it can diffuse laterally to gradually stain the cell membrane of the entire cell, and it is very stable after staining, the staining enhancer accelerates the cell staining speed, makes the fluorescent staining brighter, and the staining background is lower, the cell preservative is used to preserve negative cells and positive cells after staining, and obtains circulating tumor cell sorting quality control products, the quality control product cells are preserved in the cell preservative, the buffer is used to adjust and stabilize the acid-base environment of the solution, and glucose provides energy for cell survival. The positive cells in the quality control product are preserved after staining, and countable cells of the labeled positive cells can be directly provided in the process of cell sorting, and the sorting rate can be directly calculated to avoid the next step of antibody staining and identification, thereby improving the sorting efficiency and avoiding the waste of antibodies.
[0065] The present application also provides a method for preparing a circulating tumor cell sorting quality control product. In this embodiment, the method for preparing a circulating tumor cell sorting quality control product comprises:
[0066] Step S10, staining the positive quality control cells with a cell labeling reagent, and storing the stained positive quality control cells in a cell preservative;
[0067] The positive quality control cells used in this embodiment can be A431 cells. The staining step may include: collecting the digested positive quality control cells, incubating with a cell labeling reagent for 30 minutes, and then washing with PBS three times. It is understandable that the cell labeling reagent includes different components. The lipophilic dye can be first dissolved in a DMSO solvent to prepare a mother solution of a certain concentration. The staining enhancer is also dissolved in the DMSO solvent and prepared to the same concentration as the lipophilic dye. The stained positive cells are placed in a cell preservative and stored under certain temperature conditions. The stained positive quality control cells are stored in a cell preservative at a storage temperature of 0-4°C, and the storage time for maintaining cell activity is at least 1 month. Most of the current quality control cells are stored at a temperature below -20°C, which is not conducive to the preservation of cell activity and affects the sorting efficiency. This embodiment can be stored at a higher temperature, which is beneficial for maintaining cell activity and improving sorting efficiency.
[0068] Optionally, the proportion of the buffer in the cell preservative is 55%, and the proportion of glucose is 45%. The buffer and glucose maintain an appropriate proportion to ensure that the energy provided by the glucose can maintain the activity of the cells and the acid-base environment of the cells is appropriate.
[0069] Step S20, taking the negative quality control cells and placing them in a cell preservative;
[0070] The negative quality control cells used in this embodiment can be ball-1 cells. Like the positive quality control cells after staining, the negative quality control cells are placed in a cell preservative and stored for a certain period of time.
[0071] Step S30, taking the stored negative quality control cells and the positive quality control cells and mixing them to obtain circulating tumor cell sorting quality control products.
[0072] In one possible implementation, take 1x10 6 1000 BALL-1 cells and 1000 A431 cells were mixed to prepare a CTC sorting quality control cell.
[0073] In this embodiment, positive cells are first stained with a cell labeling reagent, and then negative cells and stained positive cells are stored in a cell preservative to prepare quality control products. The quality control cells can be stored at a higher temperature for a certain period of time to maintain the activity of the cells. When cell sorting is performed, positive countable cells can be directly provided, thereby avoiding the waste of antibodies in the antibody staining and identification steps and improving the sorting efficiency.
[0074] The present application also provides a cell sorting method, comprising:
[0075] Step A10, incubating the quality control cells obtained by the preparation method described in the above embodiment with the immunomagnetic beads at 4° C. for 2 h;
[0076] Step A20, according to the CTC sorting procedure, take the incubated magnetic beads and quality control cells for sorting on the machine, collect the obtained magnetic beads after sorting, prepare slides, view and count under immunofluorescence, and obtain the cell sorting rate.
[0077] Since the positive cells in the quality control cells have been stained, the sorting and counting steps can be carried out directly, and the subsequent antibody staining and identification can be avoided, thereby improving the sorting efficiency.
[0078] Example 1
[0079] DIO was dissolved in DMSO solvent to prepare a 30 μM stock solution. The staining enhancer Triton X-100 and polyethylene glycol were also used to prepare a 30 μM solution. The 30 μM DIO stock solution was used to prepare a 1 μM DIO dilution solution, which was mixed with the staining enhancer at the same concentration to prepare a cell labeling reagent and stain A431 cells.
[0080] Figure 1 This is a fluorescence image of cell nuclei after staining with a cell labeling reagent prepared with 1 μM DIO. Figure 2 Fluorescence images of cell labeling reagents prepared with 1 μM DIO. Figure 3 This is a cell morphology image after staining with a cell labeling reagent prepared with 1 μM DIO. Figures 1 to 3 , the cells in the field of view are basically stained with cell labeling reagents and show specific colors under fluorescence.
[0081] Example 2
[0082] DIO was dissolved in DMSO solvent to prepare a 30 μM stock solution. The staining enhancer Triton X-100 and polyethylene glycol were also used to prepare a 30 μM solution. The 30 μM DIO stock solution was used to prepare a 10 μM DIO dilution solution, which was mixed with the staining enhancer at the same concentration to prepare a cell labeling reagent and stain A431 cells.
[0083] Figure 4 This is a fluorescence image of cell nuclei after staining with a cell labeling reagent prepared with 10 μM DIO. Figure 5 Fluorescence images of cell labeling reagents prepared with 10 μM DIO. Figure 6 This is a cell morphology image after staining with a cell labeling reagent prepared with 10 μM DIO. Figures 4 to 6 , the cells in the field of view are basically stained with cell labeling reagents and show specific colors under fluorescence.
[0084] Example 3
[0085] DIO was dissolved in DMSO solvent to prepare a 30 μM stock solution. The staining enhancer Triton X-100 and polyethylene glycol were also used to prepare a 30 μM solution. The 30 μM DIO stock solution was used to prepare a 30 μM DIO dilution solution, which was mixed with the same concentration of staining enhancer to prepare a cell labeling reagent and stain A431 cells.
[0086] The cell preservative is prepared using citrate buffer and glucose, wherein the citrate buffer accounts for 55%, the glucose accounts for 45%, and the ratio of citric acid to sodium citrate in the citrate buffer is 1:1.
[0087] Figure 7 This is a fluorescence image of cell nuclei after staining with a cell labeling reagent prepared with 30 μM DIO. Figure 8 Fluorescence images of cell labeling reagents prepared with 30 μM DIO. Fig. 9 This is a cell morphology image after staining with a cell labeling reagent prepared with 30 μM DIO. Figures 7 to 9 , the cells in the field of view are basically stained with cell labeling reagents and show specific colors under fluorescence.
[0088] The stained A431 cells were stored in a cell preservative for one month at 4°C, and samples were taken on the 7th, 15th, and 30th days to observe the fluorescence and cell morphology under a fluorescence microscope. The ball-1 cells were also stored in a cell preservative for one month at 4°C, and samples were taken on the 7th, 15th, and 30th days to observe the fluorescent cell morphology under a fluorescence microscope.
[0089] Fig.10 This is a fluorescence image of cell nuclei of A431 cells stored in cell preservative at 4°C for 7 days.
[0090] Fig.11 This is a fluorescence image of cell labeling reagents for A431 cells stored in cell preservative at 4°C for 7 days.
[0091] Fig.12 The cell morphology of A431 cells stored in cell preservative at 4°C for 7 days. Fig.13 This is a fluorescence image of cell nuclei of A431 cells stored in cell preservative at 4°C for 15 days. Fig.14 This is a fluorescence image of cell labeling reagents for A431 cells stored in cell preservative at 4°C for 15 days. Fig.15 The cell morphology of A431 cells stored in cell preservative at 4°C for 15 days. Fig.16 This is a fluorescence image of cell nuclei of A431 cells stored in cell preservative at 4°C for 30 days. Fig.17This is a fluorescence image of cell labeling reagents for A431 cells stored in cell preservative at 4°C for 30 days. Fig.18 This is a cell morphology diagram of A431 cells stored in a cell preservative at 4°C for 30 days. Figure 10-18 The stained A431 cells can maintain the stained state and cell activity well within the storage time of 7 days and 15 days. Although the cell activity is lost to a certain extent after storage for 30 days, the stained state can still be maintained.
[0092] Fig.19 The cell morphology of ball-1 cells stored in cell preservative at 4°C for 7 days. Fig. 20 This is a fluorescence image of cell nuclei of ball-1 cells stored in cell preservative at 4°C for 7 days. Fig.21 The cell morphology of ball-1 cells stored in cell preservative at 4°C for 15 days. Fig. 22 This is a fluorescence image of cell nuclei of ball-1 cells stored in cell preservative at 4°C for 15 days. Fig.23 The cell morphology of ball-1 cells stored in cell preservative at 4°C for 30 days. Fig.24 This is a fluorescence image of the cell nucleus of ball-1 cells stored in a cell preservative at 4°C for 30 days. Figure 19-24 It can be found that ball-1 cells also show a trend of gradually decreasing activity.
[0093] The preserved cells were tested for circulating tumor cell sorting quality control products.
[0094] Quality control product preparation process: negative ball-1 cells and positive A431 cells were selected, cultured and collected respectively, wherein A431 cells were stained with a cell marker reagent according to the above conditions and preserved with a cell preservative. After ball-1 cells were collected, they were preserved with a cell preservative.
[0095] CTC sorting quality control process: Take the 1x10 6 1000 ball-1 cells and 1000 A431 cells were mixed, which was a CTC sorting quality control cell. The quality control cells were incubated with immunomagnetic beads matching the sorting system at 4 degrees for 2 hours. According to the CTC sorting procedure, the incubated magnetic beads and quality control cells were sorted on the machine. After the sorting was completed, the obtained magnetic beads were collected and sliced, and then viewed and counted under immunofluorescence to obtain the cell sorting rate.
[0096] Fig.25 This is the fluorescence image of the cell nuclei of the quality control product for cell sorting on the 7th day of storage. Fig.26 This is a fluorescence image of cell labeling reagents used for cell sorting on the 7th day of storage. Fig.25 and Fig.26 In the figure, the red box with blue nuclear markers but no green fluorescent markers indicates ball-1 cells. Overall, ball-1 cells will be sorted out in very small quantities, which is a normal phenomenon. The cell sorting rate on day 7 was calculated to be 80%.
[0097] Fig. 27 This is the fluorescence image of the cell nuclei of the quality control product for cell sorting on the 15th day of storage. Fig.28 This is a fluorescence image of the cell labeling reagent for cell sorting on the 15th day of storage. The cell sorting rate on the 15th day was calculated to be 78.5%.
[0098] Fig.29 This is the fluorescence image of the cell nuclei of the quality control product for cell sorting on the 30th day of storage. Fig.30 The fluorescence image of the cell labeling reagent for cell sorting on the 30th day of storage was obtained. The cell sorting rate on the 30th day was calculated to be 78%.
[0099] The sorting rates of the above cell sorting quality control products at days 7, 15 and 30 respectively meet the cell sorting rate requirements of Tianshuo cell sorters on the market.
[0100] The serial numbers of the above-mentioned embodiments of the present application are for description only and do not represent the advantages or disadvantages of the embodiments.
[0101] The above are only preferred embodiments of the present application, and are not intended to limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the present application specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.
Claims
1. A kit for preparing a circulating tumor cell sorting quality control product, characterized in that: include: Independently existing cell labeling reagent and cell preservative, the cell labeling reagent comprises DMSO solvent, lipophilic dye and staining enhancer, and the cell preservative comprises buffer and glucose.
2. The kit for preparing a circulating tumor cell separation quality control product according to claim 1, characterized in that: The lipophilic dyes include lipophilic carbocyanine dyes, lipophilic porphyrin compounds, lipophilic polycyclic aromatic hydrocarbons, and lipophilic phthalocyanine compounds.
3. The kit for preparing a circulating tumor cell sorting quality control product according to claim 1, characterized in that: The staining enhancers include surfactants and pegylation agents.
4. The kit for preparing a circulating tumor cell separation quality control product according to claim 1, characterized in that: The buffer includes citrate buffer, phosphate buffer, HEPES buffer, Tris buffer or MOPS buffer.
5. The kit for preparing a circulating tumor cell separation quality control product according to claim 4, characterized in that: The buffer is a citrate buffer, and the ratio of citric acid to sodium citrate in the buffer is 1:1-1:
3.
6. A method for preparing a circulating tumor cell sorting quality control product, characterized in that: The method is prepared using a kit for preparing a circulating tumor cell sorting quality control product according to any one of claims 1 to 5, the method comprising: The positive quality control cells are stained with a cell labeling reagent, and the stained positive quality control cells are stored in a cell preservative; Take negative quality control cells and store them in cell preservative; The stored negative quality control cells and positive quality control cells were mixed to obtain circulating tumor cell sorting quality control products.
7. The method for preparing a circulating tumor cell sorting quality control product according to claim 6, characterized in that: The buffer solution accounts for 55% of the cell preservative, and the glucose accounts for 45%.
8. The method for preparing a circulating tumor cell sorting quality control product according to claim 6, characterized in that: The step of using the cell labeling reagent to stain positive quality control cells comprises: The digested positive quality control cells were collected, incubated with cell labeling reagent for 30 min, and then washed three times with PBS.
9. The method for preparing a circulating tumor cell sorting quality control product according to claim 6, characterized in that: The stained positive quality control cells are stored in the cell preservative at a temperature of 0-4° C., and the storage time for maintaining cell activity is at least 1 month.
10. A cell sorting method, characterized in that: The method comprises: Incubate the quality control cells obtained by the preparation method according to any one of claims 6 to 9 with the immunomagnetic beads at 4° C. for 2 h; According to the CTC sorting procedure, the incubated magnetic beads and quality control cells are taken for sorting on the machine. After the sorting is completed, the obtained magnetic beads are collected and slices are made. They are viewed and counted under immunofluorescence to obtain the cell sorting rate.