CN111394416A - Cell liquid phase staining identification method with extremely small cell amount - Google Patents
Cell liquid phase staining identification method with extremely small cell amount Download PDFInfo
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Abstract
The invention provides a cell liquid phase staining identification method with extremely small cell amount, which comprises the following steps: (1) adding a cell fixing solution into the cells in a non-dry state for cell fixation; (2) after cleaning the cells, adding a cell staining solution to stain the cells; the cell staining solution comprises a fluorescent antibody staining solution; (3) staining the cells by using bright field light staining solution; (4) after the cells are washed, the cells are suspended and placed under a microscope for observation under fluorescence and bright field light, and various fluorescence and bright field light images are obtained at the same time. The invention can dye and image the cells by double light sources of fluorescent antibody and bright field optical cell morphology on the premise of no drying and no sealing, and can obtain high-quality cell fluorescent marker and bright field optical morphology imaging; the method is simple and easy to implement, has low cost, is beneficial to the classification of cells and the subsequent analysis of molecular level, and can obtain more comprehensive cell marker information.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a cell liquid phase staining identification method with few cells.
Background
In recent years, the liquid biopsy technology has the advantages of being used for tumor detection and analysis, solving a plurality of limitations on operation pathology and tissue puncture pathology, improving the sensitivity of early detection and advancing the early detection time by 3-5 years, and playing more and more important roles in the fields of clinic, scientific research, pharmacy and biotechnology. The detection of circulating tumor cells, one of the most important markers in liquid biopsy, has important and indispensable functions in counting and gene analysis, such as early detection, diagnosis, disease tracking, recurrence detection, etc., due to the advantages of the liquid biopsy markers that can represent the information of primary and diffuse tumor foci most. However, circulating tumor cells are rare cells in blood, and the number of cells obtained by enrichment and capture is small, so that the number of cells is limited in exerting the advantages of gene detection. The current method on the market is based on the capture of target cells, centrifugal on the slide glass, immunofluorescence identification, then single tumor cells by laser picking method collection, its DNA/RNA extraction, gene analysis. The method has the problems that the integrity of single cells cannot be guaranteed by the collected single cells, so that the subsequent gene analysis is caused by the technical operation that the original gene of the tumor cells is lost or the integrity of the cells is lacked when the cells are collected, no judgment is made, and when a plurality of cells are subjected to subsequent extraction of DNA/RNA and the like, main gene components are easily lost, so that the result loses the accuracy, and a lot of confusion in clinical application is caused. The existing dyeing method in the market mostly depends on dyeing on a glass slide after drying, is difficult to implement in a liquid phase, and has no method capable of simultaneously carrying out single cell fluorescence and bright field light, and meanwhile, when the single cell liquid phase fluorescence is dyed, the method has the difficulty of insufficient signals, so that no proper technology and product exist in the market.
Disclosure of Invention
In view of the disadvantages of the prior art, the present invention aims to provide a method for identifying a cell with a very small cell amount by liquid phase staining, which comprises the following steps:
(1) adding a cell fixing solution into the cells in a non-dry state for cell fixation;
(2) after cleaning the cells, adding a cell staining solution to stain the cells; the cell staining solution comprises a luminous antibody staining solution;
(3) staining the cells by using bright field light staining solution;
(4) after the cells are washed, the cells are suspended in a buffer solution and placed under a microscope, and the liquid phase is observed under fluorescence and bright field light;
the meaning of the minimal cell mass is: single cells or a limited number of cells that can be spread out sufficiently in the use vessel. For example, the limited number of cells is: in 8 square centimeter culture dish groove, the cell number is not higher than 1000.
Wherein, the luminous antibody staining solution in the step (2) is an antibody staining solution with excited luminescence (such as fluorescence) or an antibody staining solution with natural luminescence (without excited luminescence). The added staining solution of the luminescent antibody was used for protein marker observation.
In the step (3), the added bright field light staining solution is used for cell morphology observation. For those skilled in the art, any staining solution that allows observation of cell morphology in bright field can be used as the bright field light staining solution.
The cells include at least one of tumor cells, stem cells, and rare cells such as specific immune cells in body fluids and solutions. It is to be understood that the cells of the present invention are not limited to the above-exemplified cells.
The method for obtaining the cells in the non-dry state comprises the following steps: after cell culture, after cell separation (including cells obtained by separating magnetic beads, the magnetic beads are not required to be specially removed), cells are biopsied from tissues or cells in tissue cell suspension, liquid is removed but not dried, and the cells are kept wet and can be resuspended in cell buffer. It is to be understood that the method of obtaining cells in a fischer-tropsch dried state according to the invention is not limited to the above exemplified methods. It should be understood that any method that allows the cells to be humidified is within the scope of the present invention.
The fluorescent antibody staining solution comprises a primary fluorescent labeled antibody and/or a secondary fluorescent labeled antibody corresponding to the primary antibody.
When the cell staining of the step (2) is performed, the cell staining solution is used for performing cell staining at least once.
When the cell staining is carried out in the step (2), the temperature for incubating the cells is room temperature or 37 ℃.
When at least one cell staining is carried out, the staining effect can be enhanced, and the staining frequency depends on the size of the antigen and the requirement.
In a preferred embodiment of the present invention, in the step (2), after the completion of the cell staining, the following steps are further performed: adding a fluorescence-labeled antibody signal enhancement solution to perform fluorescence signal amplification on the primary fluorescence-labeled antibody and/or the secondary fluorescence antibody; the antibody signal enhancement solution comprises an amplification signal antibody which can be combined with a primary fluorescence labeling antibody and/or a secondary fluorescence antibody. This makes the cell observation clearer.
As an alternative embodiment of the present invention, the cell staining solution further comprises a cell preservation solution.
The primary fluorescence labeled antibody comprises a keratin primary fluorescence labeled antibody or a CD45 primary fluorescence labeled antibody. It is to be understood that the primary fluorescently labeled antibody of the present invention is not limited to the above-exemplified range.
In a preferred embodiment of the present invention, the temperature of cell incubation in the step (2) is room temperature. Preferably, the incubation time of the cells is 5-60 min each time the cells are stained.
As a preferred technical scheme of the invention, the cell staining solution comprises the following components in every 100 ul: 20ul of a keratin primary antibody, 2ul of a blood cell-recognizing primary antibody, 2.5ul of a blood cell-recognizing secondary antibody labeled with Alexa Fluor568, 2.5ul of a keratin-recognizing secondary antibody, and the balance of a phosphate buffer containing 1% BSA.
The invention has the beneficial effects that:
the invention can dye the fluorescent antibody and bright field optical cell morphology without drying and sealing the cell, and obtain high quality cell fluorescent marker imaging. In the prior art, cells marked by magnetic beads are always centrifuged on a glass slide after the magnetic beads are removed, and then are stained, so that the first-step marker of antibody magnetic beads is lost. The invention not only reduces the step of magnetic bead processing and adds a marker, but also directly dyes in liquid phase, thereby saving cell rupture and loss caused by centrifugation; in the liquid phase, cells are most easily and completely separated and picked, and pure target cells are obtained, so that the method is convenient for accurate gene analysis.
The method is simple and easy to implement, low in cost, beneficial to cell classification and subsequent molecular level analysis, and wide in market application prospect.
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FIG. 1 is a fluorescent and bright field light imaging chart of MCF7 breast cancer cell line obtained in example 1 of the present invention, wherein the parts from left to right in the chart are respectively a bright field chart, a DAPI staining chart, a CK staining chart, a CD45 staining chart and a staining overlay chart (DAPI staining, CK staining and CD45 staining overlay)
FIG. 2 is a photograph of fluorescent images and bright field light images of leukocytes of a parallel experimental group according to example 1 of the present invention, in which the parts from left to right are a bright field image, a DAPI staining image, a CK staining image, a CD45 staining image and a staining overlay image (DAPI staining, CK staining and CD45 staining overlay);
fig. 3 is a fluorescence and bright field light imaging diagram of lung cancer circulating tumor cells obtained in example 2 of the present invention, in which the left part to the right part of the diagram is a bright field diagram, a DAPI staining diagram, a CK staining diagram, a CD45 staining diagram and a staining overlay diagram (DAPI staining, CK staining and CD45 staining overlay);
FIG. 4 is a photograph of fluorescence and light field images of leukocytes from a parallel group in example 2 of the present invention, wherein the left part to the right part of the photograph is a bright field image, a DAPI staining image, a CK staining image, a CD45 staining image and a staining overlay image (DAPI staining, CK staining and CD45 staining overlay);
Detailed Description
The present invention is described in detail below by way of examples, and it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
Example 1
1. Raw materials and instruments: MCF7 breast cancer cell line, magnetic capture instrument (Yichangmeiguan Siligo Life technologies Co., Ltd.), sample diluent (Yichangmeiguan Siligo Life technologies Co., Ltd.), and cell staining solution.
The formulation of the cell staining solution is shown in Table 1
TABLE 1
| Components | Volume of |
| Cell binding solution 3 | 20ul |
| Cell binding solution 4 | 2ul |
| Cell staining solution 1 | 2.5ul |
| Cell staining solution 2 | 2.5ul |
| Cell preservation solution | Make up to 100ul |
Note: in table 1, cell stain 1 is a secondary antibody that recognizes a primary antibody of blood cells, and is labeled with fluorescent AlexaFluor 568; the cell staining solution 2 is a secondary antibody for recognizing a keratin primary antibody and is marked by fluorescent Alexa Fluor 488; the cell binding solution 3 is various keratin primary antibodies; the cell binding solution 4 is a primary antibody for recognizing blood cells; the cell preservation solution was phosphate buffer containing 1% BSA.
2. The experimental steps are as follows:
(1) dispersing the cultured MCF7 breast cancer cell line cells, and then separating the suspended cells together; adding a certain amount of cells into the blood sample, and placing the blood sample into a small culture dish;
(2) enriching, separating and extracting cells by using a magnetic capture instrument, and releasing and preserving the cells into a new culture dish groove filled with clean cell buffer solution;
(3) after the liquid part is sucked, under the condition of no drying, 90ul of sample diluent is dripped to the center of the release groove, and the cells are kept standing for 10min at room temperature;
(4) dripping 80ul cell binding solution 2 (cell buffer solution containing 1% BSA) to the center of the release tank, and standing the cells at room temperature for 3 min; slowly sucking and discarding the solution along the hole wall;
(5) dropping 100ul of cell staining solution in the table 1 to the center of the release tank, placing the six-hole cell culture plate on a horizontal shaking table, and incubating at room temperature at a speed of 50r/min for 60 min; after the incubation is finished, continuously dripping 90ul of cell staining solution 3 to the central position of the release groove, and incubating for 5min at room temperature;
(6) after incubation, sucking up liquid from the edge of the hole, and slowly adding 1ml of cell resuspension (phosphate buffer) into the release tank along the hole wall;
(7) and directly finishing microscopic observation after dyeing is finished: the container is placed under a microscope for observing the cell characteristics in the liquid phase under fluorescence and bright field light for identification, separation and imaging, and the results are shown in fig. 1.
Staining was performed using individual leukocytes as a parallel experimental group, referring to the staining procedure described above, and the results are shown in fig. 2.
Example 2
1. Raw materials and instruments: circulating tumor cells in blood of a lung cancer patient, a magnetic capture instrument (Yichangmeiguan silicon valley, Life technologies Co., Ltd.), a sample diluent (Yichangmeiguan silicon valley, Life technologies Co., Ltd.), and a cell staining solution (the formula is shown in Table 1 in example 1).
2. The experimental steps are as follows:
(1) releasing and preserving lung cancer circulating tumor cells captured by a magnetic capture instrument into a new culture dish groove filled with clean cell buffer solution (phosphate buffer solution);
(2) after the liquid part is sucked, under the condition of no drying, 90ul of sample diluent is dripped to the center of the release groove, and the cells are kept standing for 10min at room temperature;
(3) dripping 80ul cell binding solution 2 (cell buffer solution containing 1% BSA) to the center of the release tank, and standing the cells at room temperature for 3 min; slowly sucking and discarding the solution along the hole wall;
(4) dripping 100ul of cell staining solution to the center of the release groove, placing the six-hole cell culture plate on a horizontal shaking table, and incubating at room temperature at the speed of 50r/min for 60 min; after the incubation is finished, continuously dripping 90ul of cell staining solution 3 to the central position of the release groove, and incubating for 5min at room temperature;
(5) after incubation, sucking up liquid from the edge of the hole, and slowly adding 1ml of cell resuspension (phosphate buffer) into the release tank along the hole wall;
(6) and directly finishing microscopic observation after dyeing is finished: the container was placed under a microscope for fluorescence and bright field light observation of cell features in liquid phase for identification, isolation and imaging, the results are shown in fig. 3.
Staining was performed using individual leukocytes as a parallel experimental group, referring to the staining procedure described above, and the results are shown in fig. 4.
Example 3
1. Raw materials and instruments: circulating tumor cells of breast cancer patients, a magnetic capture instrument (Yichangmeiguan silicon valley Biotech Co., Ltd.), a sample diluent (Yichangmeiguan silicon valley Biotech Co., Ltd.), and a cell staining solution (the formula is shown in Table 1 in example 1).
2. The experimental steps are as follows:
(1) releasing and preserving the circulating tumor cells of the breast cancer patient captured by the magnetic capture instrument into a new culture dish groove filled with clean cell buffer solution (phosphate buffer solution);
(2) after the liquid part is sucked, under the condition of no drying, 90ul of sample diluent is dripped to the center of the release groove, and the cells are kept standing for 10min at room temperature;
(3) dripping 80ul cell binding solution 2 (cell buffer solution containing 1% BSA) to the center of the release tank, and standing the cells at room temperature for 3 min; slowly sucking and discarding the solution along the hole wall;
(4) dripping 100ul of cell staining solution to the center of the release groove, placing the six-hole cell culture plate on a horizontal shaking table, and incubating at room temperature at the speed of 50r/min for 60 min; after the incubation is finished, continuously dripping 90ul of cell staining solution 3 to the central position of the release groove, and incubating for 5min at room temperature;
(5) after incubation, sucking up liquid from the edge of the hole, and slowly adding 1ml of cell resuspension (phosphate buffer) into the release tank along the hole wall;
(6) and directly finishing microscopic observation after dyeing is finished: placing the container under a microscope to observe the cell characteristics under a liquid phase under fluorescence and bright field light for identification, separation and imaging;
(7) EpCAM magnetic bead positive, fluorescent immune repeat identified positive tumor cells, single or total collection, for single or small pure tumor cell gene analysis (reference method: Deng et al. BMC Cancer 2014 method).
And (3) analysis results:
PCR of exon 9 of single cell PIK3CA of 30 MCF7 identified as tumor cells, 100% successfully obtained amplified fragments, and 100% detected mutations; among the CTCs of randomly selected over 300 different breast cancer patients, 100% amplified PCR fragments of exon 9, 30% of which contained the PIK3CA mutation.
Claims (10)
1. A method for identifying a cell stain in a liquid state with a very small cell amount, comprising the steps of:
(1) adding a cell fixing solution into the cells in a non-dry state for cell fixation;
(2) after cleaning the cells, adding a cell staining solution to stain the cells; the cell staining solution comprises a luminous antibody staining solution;
(3) staining the cells by using bright field light staining solution;
(4) after the cells are washed, the cells are suspended in a buffer solution and placed under a microscope, and the liquid phase is observed under fluorescence and bright field light;
the meaning of the minimal cell mass is: single cells or a limited number of cells that can be spread out sufficiently in the use vessel.
2. The staining method of claim 1 wherein the cells comprise at least one of tumor cells, stem cells and immune cells in body fluids and solutions.
3. The staining method according to claim 1, wherein the method of obtaining the cells in a non-dried state comprises: after cell culture, cell isolation, tissue biopsy or tissue cell suspension cells, liquid removed but not dried, cells kept wet and can be resuspended in cell buffer.
4. The staining method according to claim 1, wherein the fluorescent antibody staining solution comprises a primary fluorescent-labeled antibody and/or a secondary fluorescent-labeled antibody corresponding to the primary antibody.
5. The staining method according to claim 1, wherein the cell staining of step (2) is performed by at least one cell staining using the cell staining solution.
6. The staining method according to claim 1, wherein the cell staining solution further comprises a cell preservation solution, and/or the cell staining is performed in step (2) at room temperature or 37 ℃.
7. The staining method of claim 4, wherein the primary fluorescently labeled antibody comprises a keratin primary fluorescently labeled antibody or a CD45 primary fluorescently labeled antibody.
8. The staining method according to claim 1, wherein in the step (2), after the completion of the cell staining, the following steps are further performed: adding a fluorescence-labeled antibody signal enhancement solution to perform fluorescence signal amplification on the primary fluorescence-labeled antibody and/or the secondary fluorescence antibody; the antibody signal enhancement solution comprises an amplification signal antibody which can be combined with a primary fluorescence labeling antibody and/or a secondary fluorescence antibody.
9. The staining method according to claim 6, wherein the cells are incubated for 5 to 60min per staining.
10. The staining method according to claim 1, wherein the composition of the staining solution for every 100ul of the cells comprises: 20ul of a keratin primary antibody, 2ul of a blood cell-recognizing primary antibody, 2.5ul of a blood cell-recognizing secondary antibody labeled with Alexa Fluor568, 2.5ul of a keratin primary antibody-recognizing secondary antibody labeled with Alexa Fuor488, and the balance of 1% BSA in phosphate buffered saline.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104833805A (en) * | 2015-03-09 | 2015-08-12 | 武汉格蓝丽富科技有限公司 | Circulating tumor cell detection and identification kit and application thereof |
| CN107402296A (en) * | 2017-08-22 | 2017-11-28 | 亚能生物技术（深圳）有限公司 | The immunofluorescence dyeing and interpretation method of a kind of circulating tumor cell |
| CN109752530A (en) * | 2019-01-31 | 2019-05-14 | 宜昌美光硅谷生命科技股份有限公司 | A kind of liquid phase cellular immunity identification method and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104833805A (en) * | 2015-03-09 | 2015-08-12 | 武汉格蓝丽富科技有限公司 | Circulating tumor cell detection and identification kit and application thereof |
| CN107402296A (en) * | 2017-08-22 | 2017-11-28 | 亚能生物技术（深圳）有限公司 | The immunofluorescence dyeing and interpretation method of a kind of circulating tumor cell |
| CN109752530A (en) * | 2019-01-31 | 2019-05-14 | 宜昌美光硅谷生命科技股份有限公司 | A kind of liquid phase cellular immunity identification method and its application |
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