CN115201473A - Kit for detecting NETs, preparation method and detection method thereof - Google Patents
Kit for detecting NETs, preparation method and detection method thereof Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The invention discloses a kit for detecting NETs, a preparation method and a detection method thereof, wherein the kit comprises hemolysin and a mixed fluorescent antibody; wherein the mixed fluorescent antibody comprises the following combinations: the combination is as follows: histone H3 fluorescent antibody and MPO fluorescent antibody; or a combination of two: histone H3 fluorescent antibody, MPO fluorescent antibody, and CD66 fluorescent antibody; or combining three: histone H3 fluorescent antibody, MPO fluorescent antibody and DNA double-chain specific fluorescent dye; and (4) combining: histone H3 fluorescent antibody, MPO fluorescent antibody, CD66 fluorescent antibody, and DNA double strand specific fluorescent dye. Can directly detect a whole blood sample, has strong specificity of a detection result, and is suitable for detecting a large sample.
Description
Technical Field
The invention belongs to the technical field of medical detection, and particularly relates to a kit for detecting NETs, a preparation method and a detection method thereof.
Background
Neutrophils are one of the important components of the natural immunity of the human body. When the neutrophil is subjected to stimuli such as infection and inflammation, intracellular chromatin, nucleoprotein, granulin and the like are released to the outside of the cell to form extracellular traps (NETs). Neutrophil extracellular traps, namely NETs (also called neutrophil extracellular trapping NETs), play an important role in defending against pathogen infection, but too many extracellular traps can cause damage to human bodies, the NETs are obviously increased in various pathological states, such as severe infection, cardiovascular and cerebrovascular diseases, tumors, autoimmune diseases and the like, the content of the NETs in the bodies of patients can be detected, the disease state can be prompted, and a therapeutic scheme can be guided.
The detection of NETs has important clinical value, but a detection method directly aiming at clinical specimens is lacked at present.
CN 108362871A and CN 113238050A disclose two different net detection methods, but there are common problems: 1. both methods are used for in vitro culture and stimulated neutrophils, and cannot be used for directly detecting a whole blood sample. 2. In both methods, only one specific antibody is combined with one DNA fluorescent dye for detection, and the antibody is not selected, so that the method is easily interfered and the specificity of the detection result is insufficient. 3. The operation process is complicated, and the method is not suitable for the use requirement of large sample detection.
Disclosure of Invention
In view of at least one of the above technical problems, the present invention aims to provide a kit for detecting NETs, a preparation method thereof and a detection method thereof, which can directly detect a whole blood sample, have strong specificity of detection results, and are suitable for large sample detection.
The technical scheme of the invention is as follows:
one of the purposes of the invention is to provide a kit for detecting NETs, which comprises hemolysin and a mixed fluorescent antibody; wherein the mixed fluorescent antibody comprises the following combinations:
the combination is as follows: histone H3 fluorescent antibody and MPO fluorescent antibody; or
Combining two: histone H3 fluorescent antibody, MPO fluorescent antibody, and CD66 fluorescent antibody; or
Combining three components: histone H3 fluorescent antibody, MPO fluorescent antibody and DNA double-chain specific fluorescent dye;
and (4) combining: histone H3 fluorescent antibody, MPO fluorescent antibody, CD66 fluorescent antibody, and DNA double strand specific fluorescent dye.
Preferably, the histone H3 antibody is an anti-citrulline modified histone H3 mouse anti-human monoclonal antibody Ab-CitH3, and the antigen sequence aimed at by the antibody is an epitope of citrulline modified C-terminal 1 st-30 th amino acid sequence.
Preferably, the histone H3 fluorescent antibody, the MPO fluorescent antibody and the CD66 antibody are respectively marked with fluorescent dyes with different emission wavelengths.
Preferably, in the kit of any combination, the ratio of each antibody is 1.
Preferably, the fluorescent microspheres are used for absolute quantification, and the final concentration of the fluorescent microspheres is 10 5 -10 6 One per ml.
Another object of the present invention is to provide a method for preparing the above kit, comprising the steps of:
preparation of a storage solution:
(1) Dissolving calf serum albumin in phosphate buffer at pH =7.4 to form phosphate buffer containing 1% calf serum albumin;
(2) Adding ProClin 300 to make the final concentration of the buffer solution be 0.03%;
(3) Filtering and sterilizing to dilute the fluorescent antibody;
preparation of histone H3 fluorescent antibody and MPO fluorescent antibody:
(1) The histone H3 antibody and the MPO antibody were diluted to 1mg/ml with phosphate buffer, respectively, as 10:1, adding an activating solution, uniformly shaking, and incubating at room temperature for 10min;
(2) Respectively adding fluorescein with the same mass as the antibody, uniformly mixing, and incubating at 4 ℃ in a dark place for 12h to obtain the fluorescent antibody;
(3) Adding one tenth of the volume of the quencher respectively, mixing uniformly, and incubating for 30min at room temperature in a dark place;
(4) Respectively purifying and separating the marked fluorescent antibody by adopting a chromatographic column;
(5) Measuring the antibody titer by an agarose diffusion method, and measuring the antibody quality by a colorimetric method;
(6) Respectively diluting the separated and purified fluorescent antibody to 100 mu g/ml by using a storage solution, and storing the fluorescent antibody at 4 ℃ in a dark place;
mixing and subpackaging antibodies:
diluted histone H3 fluorescent antibody, MPO fluorescent antibody and CD66 antibody with a concentration of 100 μ g/ml were mixed at a ratio of 1:1:1 to form mixed fluorescent antibodies, and subpackaging the mixed fluorescent antibodies in brown reagent tubes, wherein each tube contains 1ml of the mixed fluorescent antibody;
split charging of hemolysin:
subpackaging hemolysin into 5ml reagent tubes;
subpackaging fluorescent dye:
dissolving Sytox Green or Sytox Orange in dimethyl sulfoxide to make the concentration be 1mM, subpackaging in brown reagent tubes with 1ml each, and storing in dark place;
packaging of the kit:
and (3) packaging the subpackaged mixed fluorescent antibody, hemolysin and fluorescent dye into a packaging box, putting a specification into the packaging box, sealing the packaging box, printing a production batch number and an effective period, and storing at 2-8 ℃ after quality inspection is qualified.
Still another object of the present invention is to provide a rapid detection method for detecting NETs using the kit prepared according to the above kit or the preparation method, comprising the steps of:
the method comprises the following steps: adding 10-30 mul of mixed fluorescent antibody and DNA double-chain specific dye into a special test tube of a flow cytometer;
step two: adding 20-100 mul of whole blood sample into the mixed fluorescent antibody, mixing the whole blood sample and the mixed fluorescent antibody evenly, and incubating the whole blood sample for 15-25 minutes in a dark place;
step three: adding 0.3-1.5ml of hemolysin into the test tube, mixing evenly, and incubating for 10-20 minutes in a dark place;
step four: adding fluorescent microspheres;
step five: and detecting by using a flow cytometer.
The invention also aims to provide another rapid detection method for detecting NETs by using the kit prepared by the kit or the preparation method, which comprises the following steps:
the method comprises the following steps: adding 10-30 mul of mixed fluorescent antibody into a special test tube of a flow cytometer;
step two: adding 20-100 mul of whole blood sample into the mixed fluorescent antibody, mixing the whole blood sample and the mixed fluorescent antibody evenly, and incubating the whole blood sample for 15-25 minutes in a dark place;
step three: adding 0.3-1.5ml of hemolysin into the test tube, lightly mixing uniformly, and incubating for 10-20 minutes in a dark place;
step four: adding 1-4ml of phosphate buffer solution for washing, centrifuging for 5 minutes at 500g, and removing supernatant;
step five: repeating the step four;
step six: adding 200 mul of phosphate buffer solution to suspend the cells;
step seven: adding fluorescent microspheres;
step eight: and detecting by using a flow cytometer.
Preferably, the final concentration of each of the mixed fluorescent antibodies in the reaction system is 2 to 30. Mu.g/ml.
Preferably, the concentration of the DNA double strand specific fluorescent dye in the reaction system is 10 nM-1. Mu.M.
Compared with the prior art, the invention has the advantages that:
the kit for detecting NETs is simple and convenient, can directly detect the neutrophil NETs (NETs) in human whole blood cells, does not need to separate the neutrophil NETs, and does not need to carry out in-vitro culture and stimulation. The specificity of the detection result is strong, the result is stable and reliable, and the method is suitable for detecting large samples.
Drawings
The invention is further described with reference to the following figures and examples:
FIG. 1 is a diagram showing the results of the kit for detecting NETs of example 1 of the present invention for detecting NETs in normal human whole blood cells;
FIG. 2 is a graph showing the results of the assay for detecting NETs in whole blood cells of an infected patient using the kit for detecting NETs according to example 1 of the present invention;
FIG. 3 is a diagram showing the results of detection of normal human NETs by the kit of the comparative example;
fig. 4 is a graph showing the results of the test of the kit of the control example for testing NETs of infected patients.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings in conjunction with the following detailed description. It is to be understood that these descriptions are only illustrative and are not intended to limit the scope of the present invention. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present invention.
Example 1
The kit for detecting NETs (neutrophils) extracellular traps comprises hemolysin, a mixed fluorescent antibody and fluorescent microspheres; wherein the mixed fluorescent antibody comprises a histone H3 fluorescent antibody, an MPO (myeloperoxidase) fluorescent antibody, a CD66 fluorescent antibody and a DNA double-strand specific fluorescent dye. The final concentration of fluorescent microspheres was 10 5 -10 6 Each/ml. The addition of a CD66 fluorescent antibody can improve the convenience of result analysis, because CD66 can be used to more conveniently gate (in flow analysis terms) the target cells to be analyzed.
According to some preferred embodiments of the invention, the histone H3 antibody is anti-citrullinated histone H3 murine anti-human monoclonal antibody Ab-CitH3 directed against an epitope of citrullinated C-terminal amino acid sequence 1-30.
According to some preferred embodiments of the present invention, the histone H3 fluorescent antibody, the MPO fluorescent antibody, and the CD66 antibody are labeled with fluorescent dyes having different emission wavelengths, respectively. Such as CitH3-FITC (fluorescein isothiocyanate), MPO-PE, and CD66-Cy5, but not limited to FITC, PE, and Cy5 labels.
According to some preferred embodiments of the invention, in any combination antibody kit, the ratio value for each antibody is 1. Preferably, the final concentration of each fluorescent antibody in the reaction system is 2 to 30. Mu.g/ml.
It should be noted that in the embodiment of the present invention, the DNA double strand specific fluorescent dye may be Sytox Green, sytox Orange or Propidium Iodide (PI), which is commonly known in the art.
Compared with the comparison documents 1 and 2 in the background art, the kit provided by the embodiment of the invention can be directly used for detecting a whole blood sample of a patient and assisting diagnosis.
Example 2
The kit for detecting NETs comprises hemolysin and a mixed fluorescent antibody. Unlike example 1, the mixed fluorescent antibody in the present example includes a histone H3 fluorescent antibody and an MPO fluorescent antibody. The rest is the same as the embodiment 1, and the description is omitted.
Example 3
The kit for detecting NETs comprises hemolysin and a mixed fluorescent antibody. Unlike example 2, the mixed fluorescent antibody in the present example includes a histone H3 fluorescent antibody, an MPO fluorescent antibody, and a DNA double strand specific fluorescent dye. The rest is the same as embodiment 2 and will not be described again.
Example 4
The kit for detecting NETs comprises hemolysin and a mixed fluorescent antibody. Unlike example 3, the mixed fluorescent antibody in the present example includes a histone H3 fluorescent antibody, an MPO fluorescent antibody, a DNA double strand specific fluorescent dye, and CD66. The rest is the same as embodiment 3 and will not be described again.
Example 5
The kit for detecting NETs comprises hemolysin and a mixed fluorescent antibody. The mixed fluorescent antibody in the embodiment of the invention comprises a histone H3 fluorescent antibody and an MPO fluorescent antibody. Unlike example 2, the kit according to the present example further includes fluorescent microspheres for absolute quantification. The rest is the same as embodiment 2 and will not be described again.
Example 6
The kit for detecting NETs comprises hemolysin and a mixed fluorescent antibody. The mixed fluorescent antibody in the embodiment of the invention comprises a histone H3 fluorescent antibody, an MPO fluorescent antibody and a DNA double-chain specific fluorescent dye. The difference from example 3 is that the kit of the present example further includes fluorescent microspheres for absolute quantification. The rest is the same as embodiment 3 and will not be described again.
Example 7
The preparation method of the kit provided by the embodiment of the invention is that the kit provided by any one of embodiments 1-6 is used for detecting NETs, and comprises the following steps:
preparation of a storage liquid:
(1) Dissolving Bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS) at pH =7.4 to form a phosphate buffered saline containing 1% bovine serum albumin;
(2) Adding externally purchased ProClin 300 to make the final concentration of the buffer solution be 0.03%;
(3) Filtering and sterilizing to dilute the fluorescent antibody;
preparation of histone H3 fluorescent antibody and MPO fluorescent antibody:
(1) The histone H3 antibody and the MPO antibody were diluted to 1mg/ml with Phosphate Buffered Saline (PBS), respectively, as 10:1, adding an activating solution, uniformly shaking, and incubating at room temperature for 10min;
(2) Respectively adding fluorescein with the same mass as the antibody, uniformly mixing, and incubating at 4 ℃ in a dark place for 12h to obtain the fluorescent antibody;
(3) Adding one tenth of the volume of the quencher respectively, mixing uniformly, and incubating for 30min at room temperature in a dark place;
(4) Respectively purifying and separating the marked fluorescent antibody by adopting a chromatographic column;
(5) Measuring the antibody titer by an agarose diffusion method, and measuring the antibody quality by a colorimetric method;
(6) Diluting the separated and purified fluorescent antibody to 100 mu g/ml with a storage solution respectively, and storing at 4 ℃ in a dark place;
mixing and subpackaging antibodies:
diluted histone H3 fluorescent antibody, MPO fluorescent antibody and CD66 antibody with a concentration of 100 μ g/ml were mixed at a ratio of 1:1:1 to form mixed fluorescent antibodies, and subpackaging the mixed fluorescent antibodies in brown reagent tubes, wherein each tube contains 1ml of the mixed fluorescent antibody;
split charging of hemolysin:
subpackaging hemolysin into 5ml reagent tubes;
subpackaging fluorescent dye:
dissolving purchased Sytox Green or Sytox Orange in dimethyl sulfoxide to make the concentration be 1mM, subpackaging in brown reagent tubes with 1ml each, and storing in dark place;
packaging of the kit:
and (3) packaging the subpackaged mixed fluorescent antibody, hemolysin and fluorescent dye into a packaging box, putting a specification into the packaging box, sealing the packaging box, printing a production batch number and an effective period, and storing at 2-8 ℃ after quality inspection is qualified.
Example 8
The kit is the kit for detecting NETs in embodiment 1 or the kit prepared by the preparation method of embodiment 7, and the detection method comprises the following steps:
the method comprises the following steps: adding 10-30 mul of mixed fluorescent antibody and DNA double-chain specific dye into a special test tube of a flow cytometer;
step two: adding 20-100 mul of whole blood sample into the mixed fluorescent antibody, mixing the whole blood sample and the mixed fluorescent antibody evenly, and incubating the whole blood sample for 15-25 minutes in a dark place;
step three: adding 0.3-1.5ml of hemolysin into the test tube, lightly mixing uniformly, and incubating for 10-20 minutes in a dark place;
step four: adding fluorescent microspheres for absolute quantification;
step five: and detecting by using a flow cytometer.
In the embodiment, the detection method does not comprise a cleaning step, so that the steps are fewer, and the time is saved.
Example 9
The detection method of the kit provided by the embodiment of the invention is the kit for detecting NETs in embodiment 1 or the kit prepared by the preparation method in embodiment 7, and is different from the detection method in embodiment 5 in that the detection method of the embodiment comprises a PBS buffer solution washing step, supernatant liquid is removed by centrifugation in the washing process, cells are precipitated at the bottom of a tube, and the cells are suspended by adding the PBS buffer solution. Specifically, the detection method comprises the following steps:
the method comprises the following steps: adding 10-30 mul of mixed fluorescent antibody into a special test tube of a flow cytometer;
step two: adding 20-100 mul of whole blood sample into the mixed fluorescent antibody, gently mixing uniformly, and incubating for 15-25 minutes in a dark place;
step three: adding 0.3-1.5ml of hemolysin into the test tube, lightly mixing uniformly, and incubating for 10-20 minutes in a dark place;
step four: adding 1-4ml Phosphate (PBS) buffer solution for washing, centrifuging for 5 minutes at 500g, and discarding the supernatant;
step five: repeating the step four;
step six: cells were suspended by adding 200. Mu.l of Phosphate (PBS) buffer;
step seven: adding fluorescent microspheres for absolute quantification;
step eight: and detecting by using a flow cytometer.
Comparative example
The difference from example 1 is that the mixed fluorescent antibody in the kit of the control example is a histone H1 fluorescent antibody, an MPO fluorescent antibody, a CD66 fluorescent antibody and a DNA double strand specific fluorescent dye.
In order to test the effect of the kit in the embodiment of the invention, the kit in the embodiment 1 of the invention and the kit in the control embodiment are respectively used for testing the effect of the neutrophil NETs of a normal person and a patient infected with the neutrophil NETs (the samples in the embodiment 1 of the invention are whole blood samples of the normal person and whole blood samples of the patient infected with the neutrophil NETs, the samples in the control embodiment are neutrophils after in vitro culture and stimulation), the test results are shown in the figures 1 to 4, the four quadrant areas in the figures are respectively represented by Q1, Q2, Q3 and Q4, the number of each quadrant is the proportion of cells, the relative quantitative result is obtained, and the qualitative result only needs to see whether the Q2 has positive cells. The results of absolute quantification were also automatically calculated by flow cytometry after addition of counting microspheres. In the embodiment of the invention, the graphs shown in fig. 1 to 4 are relative quantitative result graphs. Wherein, in FIGS. 1 and 2, Q1 represents MPO positive and H3 negative; q2 indicates that both are positive; q3 is MPO negative and H3 positive; q4 indicates that both are negative. Similarly, in FIGS. 3 and 4, Q1 indicates MPO positive and H1 negative; q2 indicates that both are positive; q3 represents MPO negative and H1 positive; q4 indicates that both are negative. As can be seen from the comparison results of fig. 1 and 3 and the comparison results of fig. 2 and 4, the kit of example 1 of the present invention has a better effect of labeling staining than the kit of the comparison example, i.e., the combination of the comparison document 1 and the comparison document 2 in the background art, and has strong specificity of the detection result, a greatly increased number of detectable cells, and a more stable and reliable detection result.
It is to be understood that the above-described embodiments of the present invention are merely illustrative of or explaining the principles of the invention and are not to be construed as limiting the invention. Therefore, any modifications, equivalents, improvements and the like which are made without departing from the spirit and scope of the present invention shall be included in the protection scope of the present invention. Further, it is intended that the appended claims cover all such variations and modifications as fall within the scope and boundaries of the appended claims or the equivalents of such scope and boundaries.
Claims (10)
1. A kit for detecting NETs is characterized by comprising hemolysin and a mixed fluorescent antibody; wherein the mixed fluorescent antibody comprises the following combinations:
the combination is as follows: histone H3 fluorescent antibody and MPO fluorescent antibody; or
Combining two: histone H3 fluorescent antibody, MPO fluorescent antibody, and CD66 fluorescent antibody; or
Combining three components: histone H3 fluorescent antibody, MPO fluorescent antibody and DNA double-chain specific fluorescent dye;
and (4) combining: histone H3 fluorescent antibody, MPO fluorescent antibody, CD66 fluorescent antibody, and DNA double strand specific fluorescent dye.
2. The kit for detecting NETs of claim 1, wherein the histone H3 antibody is an anti-citrullinated histone H3 murine anti-human monoclonal antibody Ab-CitH3, which is directed against an epitope of citrullinated C-terminal amino acid sequence 1-30.
3. The kit for detecting NETs according to claim 1, wherein the histone H3 fluorescent antibody, the MPO fluorescent antibody and the CD66 antibody are respectively labeled with fluorescent dyes with different emission wavelengths.
4. The kit for detecting NETs according to claim 1, characterized in that, in the kit of any combination, the ratio value of each antibody is 1.
5. Kit for the detection of NETs according to any one of claims 1-4, characterized in that it further comprises fluorescent microspheres for absolute quantification, the final concentration of fluorescent microspheres being 10 5 -10 6 One per ml.
6. The method for preparing a kit according to any one of claims 1 to 5, comprising the steps of:
preparation of a storage solution:
(1) Dissolving calf serum albumin in phosphate buffer at pH =7.4 to form phosphate buffer containing 1% calf serum albumin;
(2) Adding ProClin 300 to make the final concentration of the buffer solution be 0.03%;
(3) Filtering and sterilizing to dilute the fluorescent antibody;
preparation of histone H3 fluorescent antibody and MPO fluorescent antibody:
(1) The histone H3 antibody and the MPO antibody were diluted to 1mg/ml with phosphate buffer, respectively, as 10:1, adding an activating solution, uniformly shaking, and incubating at room temperature for 10min;
(2) Respectively adding fluorescein with the same mass as the antibody, uniformly mixing, and incubating at 4 ℃ in a dark place for 12h to obtain the fluorescent antibody;
(3) Adding one tenth of the volume of the quencher respectively, mixing uniformly, and incubating for 30min at room temperature in a dark place;
(4) Respectively purifying and separating the marked fluorescent antibody by adopting a chromatographic column;
(5) Measuring the antibody titer by an agarose diffusion method, and measuring the antibody quality by a colorimetric method;
(6) Diluting the separated and purified fluorescent antibody to 100 mu g/ml with a storage solution respectively, and storing at 4 ℃ in a dark place;
mixing and subpackaging the antibodies:
diluted histone H3 fluorescent antibody, MPO fluorescent antibody and CD66 antibody at a concentration of 100 μ g/ml were mixed at a ratio of 1:1:1 to form mixed fluorescent antibodies, and subpackaging the mixed fluorescent antibodies in brown reagent tubes, wherein each tube contains 1ml of the mixed fluorescent antibody;
split charging of hemolysin:
subpackaging hemolysin into 5ml reagent tubes;
subpackaging fluorescent dye:
dissolving purchased Sytox Green or Sytox Orange in dimethyl sulfoxide to make the concentration be 1mM, subpackaging in brown reagent tubes with 1ml each, and storing in dark place;
packaging of the kit:
and (3) packaging the subpackaged mixed fluorescent antibody, hemolysin and fluorescent dye into a packaging box, putting a specification into the packaging box, sealing the packaging box, printing a production batch number and an effective period, and storing at 2-8 ℃ after quality inspection is qualified.
7. A method for detecting NETs using the kit of any one of claims 1 to 5 or the kit prepared by the preparation method of claim 6, comprising the steps of:
the method comprises the following steps: adding 10-30 mul of mixed fluorescent antibody and DNA double-chain specific dye into a special test tube of a flow cytometer;
step two: adding 20-100 mul of whole blood sample into the mixed fluorescent antibody, gently mixing uniformly, and incubating for 15-25 minutes in a dark place;
step three: adding 0.3-1.5ml of hemolysin into the test tube, lightly mixing uniformly, and incubating for 10-20 minutes in a dark place;
step four: adding fluorescent microspheres;
step five: and detecting by using a flow cytometer.
8. A method for detecting NETs using the kit according to any one of claims 1 to 5 or the kit prepared by the preparation method according to claim 6, comprising the steps of:
the method comprises the following steps: adding 10-30 mul of mixed fluorescent antibody into a special test tube of a flow cytometer;
step two: adding 20-100 mul of whole blood sample into the mixed fluorescent antibody, mixing the whole blood sample and the mixed fluorescent antibody evenly, and incubating the whole blood sample for 15-25 minutes in a dark place;
step three: adding 0.3-1.5ml of hemolysin into the test tube, mixing evenly, and incubating for 10-20 minutes in a dark place;
step four: adding 1-4ml of phosphate buffer solution for washing, centrifuging for 5 minutes at 500g, and removing supernatant;
step five: repeating the step four;
step six: adding 200 mul of phosphate buffer solution to suspend the cells;
step seven: adding fluorescent microspheres;
step eight: and detecting by using a flow cytometer.
9. The detection method according to claim 7 or 8, wherein the final concentration of each of the mixed fluorescent antibodies in the reaction system is 2 to 30 μ g/ml.
10. The detection method according to claim 7 or 8, wherein the concentration of the DNA double strand-specific fluorescent dye in the reaction system is 10 nM-1. Mu.M.
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