CN112946264A - Sample pretreatment method and immune cell detection method for flow cytometry - Google Patents
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Abstract
The application relates to the technical field of biomarker detection, in particular to a sample pretreatment method and an immune cell detection method for flow cytometry. The sample pretreatment method comprises the following steps: extracting peripheral blood lymphocytes; and washing the peripheral blood lymphocytes by using a membrane breaking agent, and adding a beta-galactosidase primary antibody and a fluorescence-labeled secondary antibody for incubation treatment. According to the treatment method, the beta-galactosidase inside and outside the cells is labeled by the beta-galactosidase primary antibody and the fluorescence labeled secondary antibody, so that the beta-galactosidase of the lymphocytes can be rapidly, sensitively and accurately quantitatively detected in the subsequent flow cytometry detection, and the treatment method has a good application prospect.
Description
Technical Field
The application belongs to the technical field of biomarker detection, and particularly relates to a sample pretreatment method and an immune cell detection method for flow cytometry.
Background
The immune system is an important system for an organism to execute immune response and immune function, consists of immune organs, immune cells and immune molecules, and has the functions of identifying and removing antigenic foreign matters, coordinating with other systems of the organism and maintaining the stable environment and physiological balance in the organism together. Among them, lymphocytes are one of the most important immune cells, and mainly include T cells (T lymphocytes), B cells (B lymphocytes), and NK cells (Natural Killer cells). The immunosenescence condition of the human body can be evaluated by detecting the senescence condition of various lymphocytes.
Cellular aging is an irreversible growth cycle arrest that can be triggered by a variety of factors, including DNA damage, oxidative stress, and the like. Senescent cells retain some metabolic activity for a considerable period of time, but the expression profile of the cells for genes and proteins is greatly altered. In addition to cell cycle arrest, senescent cells generally become large and express beta-galactosidase (beta-galactosidase) with high enzymatic activity, and thus beta-galactosidase is often used as a marker for senescent cells.
At present, the detection of cell senescence usually adopts a commercial beta-galactosidase staining kit, X-Gal (5-bromo-4-chloro-3-indole-beta-D-galactoside) is taken as a substrate, a dark blue product is generated under the catalysis of senescence-specific beta-galactosidase, and cells or tissues expressing the beta-galactosidase, which turn blue, are observed under an optical microscope. Although this method of cellular senescence detection is more commonly used, it has three major drawbacks: (1) only fixed cells can be detected; (2) it is time consuming, long (>12h) staining time, and after staining it is necessary to detect the degree of senescence by counting a large number of senescent cells under a microscope; (3) because the differentiation of senescent cells from non-senescent cells is completely dependent on the subjective judgment of experimenters, certain errors exist.
Therefore, the related art is in need of improvement.
Disclosure of Invention
The application aims to provide a sample pretreatment method for flow cytometry detection and an immune cell detection method, and aims to solve the technical problems of complex detection steps, low sensitivity and low accuracy of beta-galactosidase in the existing immune cells.
In order to achieve the purpose of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides a sample pretreatment method for flow cytometry, comprising the steps of:
extracting peripheral blood lymphocytes;
and washing the peripheral blood lymphocytes by using a membrane breaking agent, and adding a beta-galactosidase primary antibody and a fluorescence-labeled secondary antibody for incubation treatment.
The sample pretreatment method for flow cytometry is a peripheral blood lymphocyte sample pretreatment method for flow cytometry, and the method comprises the steps of washing extracted peripheral blood lymphocytes with a membrane breaking agent to break membranes of the cells, adding beta-galactosidase primary antibody and a fluorescence labeled secondary antibody to incubate, so that the specific primary antibody and the specific secondary antibody label beta-galactosidase inside and outside the cells; when the treated sample is subsequently subjected to flow cytometry detection, the beta-galactosidase of the lymphocyte can be rapidly, sensitively and accurately quantitatively detected, and the application prospect is good.
In a second aspect, the present application provides a method for detecting immune cells, comprising the steps of;
providing a peripheral blood lymphocyte sample to be tested; the sample to be detected of the peripheral blood lymphocytes is a sample treated by the sample pretreatment method;
and detecting the peripheral blood lymphocyte sample to be detected by using a flow cytometer.
The immune cell detection method provided by the application is a flow detection method for beta-galactosidase of peripheral blood lymphocytes by adopting a flow cytometer, and a sample to be detected of the peripheral blood lymphocytes is a sample treated by the sample pretreatment method, and compared with the existing beta-galactosidase staining detection method, the detection method has the following remarkable advantages: (1) can be used for detecting living cells or fixed cells; (2) the operation is simple, and the sensitivity is high; (3) the flow cytometer is used for accurate quantitative analysis, so that subjective errors caused by manual counting are avoided.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows NC groups and H in experiment I2O2Total lymphocyte assay control images for the groups (micrographs of the control protocol on the left; flow assays for the protocol of the present application on the right);
FIG. 2 shows NC groups and H in experiment I2O2Statistical plots of the aging ratios for the total lymphocyte assay results for the groups (statistics for the comparative protocol on the left; statistics for the protocol of the present application on the right);
FIG. 3 shows NC groups and H in experiment two2O2T cell assay contrast plots for the groups (flow assay plot for contrast protocol on the left; flow assay plot for the protocol of the present application on the right);
FIG. 4 shows NC groups and H in experiment two2O2Statistical plots of aging ratios for the T cell test results for the groups (statistics for the comparative protocol on the left; statistics for the protocol of the present application on the right);
FIG. 5 shows NC groups and H in experiment III2O2NK cell detection contrast maps for panels (flow detection map for contrast protocol on left; flow detection map for protocol of the present application on right);
FIG. 6 shows NC groups and H in experiment III2O2Statistical figures of senescence ratios for the set of NK cell assay results (statistics for the comparative protocol on the left; statistics for the protocol of the present application on the right);
FIG. 7 shows NC groups and H in experiment four2O2B cell assay contrast maps for the groups (flow assay map for contrast protocol on the left; flow assay map for the protocol of the present application on the right);
FIG. 8 shows NC groups and H in experiment four2O2Statistical plots of the senescence ratios of the group's B cell assays (statistics for the control protocol on the left and the protocol of the present application on the right).
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present application more clearly apparent, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
The terminology used in the embodiments of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in the examples of this application and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The term "primary antibody" refers to a protein capable of specifically binding to a non-antibody antigen (specific antigen), such as β -galactosidase primary antibody refers to an antibody protein capable of specifically binding to β -galactosidase, and "secondary antibody" refers to a protein capable of binding to an antibody, i.e., an antibody to an antibody.
The first aspect of the embodiments of the present application provides a sample pretreatment method for flow cytometry, including the following steps:
s01: extracting peripheral blood lymphocytes;
s02: and washing the peripheral blood lymphocytes by using a membrane breaking agent, and adding a beta-galactosidase primary antibody and a fluorescence-labeled secondary antibody for incubation treatment.
The sample pretreatment method for flow cytometry is a peripheral blood lymphocyte sample pretreatment method for flow cytometry, and the method comprises the steps of washing extracted peripheral blood lymphocytes with a membrane breaking agent to break membranes of the cells, adding beta-galactosidase primary antibody and a fluorescence labeled secondary antibody to incubate, so that the specific primary antibody and the specific secondary antibody label beta-galactosidase inside and outside the cells; when the treated sample is subsequently subjected to flow cytometry detection, the beta-galactosidase of the lymphocyte can be rapidly, sensitively and accurately quantitatively detected, and the application prospect is good.
In some embodiments, the peripheral blood lymphocytes can be extracted by using a commonly used peripheral blood lymphocyte separation solution, and 50mL of peripheral blood is treated by using a human peripheral blood lymphocyte separation solution to separate total lymphocytes. The resulting total lymphocytes were washed with DPBS (dunaliella phosphate buffered saline) and resuspended.
In some embodiments, the membrane-breaking agent is an intracellular staining penetration wash, and specifically may be a Triton X-100 (polyethylene glycol octylphenyl ether) solution.
In some embodiments, the step of adding a primary β -galactosidase antibody and a fluorescently labeled secondary antibody for incubation comprises: and (3) firstly adding the beta-galactosidase primary antibody, standing for 40-80min, and then adding the fluorescence-labeled secondary antibody, and incubating for 15-20 min in a dark place. The incubation conditions may be such that the primary and secondary antibodies are better labeled with beta-galactosidase. Further, after the step of adding the beta-galactosidase primary antibody and the fluorescently-labeled secondary antibody for incubation treatment, washing with the membrane-breaking agent and the buffer solution in sequence is further included.
In some embodiments, the primary beta-galactosidase antibody is a primary beta-galactosidase rabbit derived antibody; the fluorescently-labeled secondary antibody is a phycoerythrin (Pphycoerythrin, PE) labeled mouse anti-rabbit immunoglobulin G (IgG) secondary antibody.
In some embodiments, before the step of washing the peripheral blood lymphocytes with the cell disrupting agent, the method further comprises: the peripheral blood lymphocytes were stained with flow antibody CD3-FITC (i.e., fluorescein isothiocyanate FITC labeled CD3 antibody), and then fixed with a fixative. This, in combination with subsequent labeling of the beta-galactosidase by the primary beta-galactosidase antibody and the fluorescently labeled secondary antibody, allows the detection of T cells in peripheral blood lymphocytes. Meanwhile, the accuracy of the T cell senescence assay of the application is further shown by comparing the flow cytometry assay directly stained with the flow antibodies CD3-FITC and CD57-PE (namely the phycoerythrin fluorescein PE-labeled CD57 antibody).
Alternatively, the peripheral blood lymphocytes were stained with flow-through antibodies CD3-FITC and CD56-PC5.5 (i.e., fluorescein PerCP-Cy5.5 labeled CD56 antibody), and then fixed with a fixative. This, in combination with subsequent labeling of the beta-galactosidase by the primary beta-galactosidase antibody and the fluorescently labeled secondary antibody, allows the detection of NK cells in peripheral blood lymphocytes. Meanwhile, the accuracy of the NK cell senescence detection of the application is further shown by comparing the flow cytometry detection directly stained by the flow antibodies CD3-FITC, CD56-PC5.5 and CD 57-PE.
Alternatively, the peripheral blood lymphocytes were stained with flow-through antibodies CD3-FITC and CD19-PC5.5 (i.e., fluorescein PerCP-Cy5.5 labeled CD19 antibody); then, fixing treatment is carried out by using a fixing agent. This, in combination with subsequent labeling of the beta-galactosidase by the primary beta-galactosidase antibody and the fluorescently labeled secondary antibody, allows the detection of B cells in peripheral blood lymphocytes. Meanwhile, the accuracy of the B cell senescence assay of the application is further shown by comparing the flow cytometric assay directly stained with flow antibodies CD3-FITC, CD19-PC5.5 and IgD-PE (namely, phycoerythrin fluorescein PE-labeled immunoglobulin D antibody).
Further, the fixing agent may be a fixing solution of paraformaldehyde, and in an embodiment, 4% paraformaldehyde may be used for fixing at room temperature (25-27 ℃).
In a second aspect, the embodiments of the present application provide a method for detecting immune cells, comprising the steps of;
e01: providing a peripheral blood lymphocyte sample to be tested; the sample to be detected of the peripheral blood lymphocytes is a sample treated by the sample pretreatment method;
e02: and detecting the peripheral blood lymphocyte sample to be detected by using a flow cytometer.
The immune cell detection method provided by the application is a flow detection method for beta-galactosidase of peripheral blood lymphocytes by adopting a flow cytometer, and a sample to be detected of the peripheral blood lymphocytes is a sample treated by the sample pretreatment method, and compared with the existing beta-galactosidase staining detection method, the detection method has the following remarkable advantages: (1) can be used for detecting living cells or fixed cells; (2) the operation is simple, and the sensitivity is high; (3) the flow cytometer is used for accurate quantitative analysis, so that subjective errors caused by manual counting are avoided.
The sample to be detected for the peripheral blood lymphocytes is a sample treated by an optimized pretreatment method for the sample of the human peripheral blood lymphocytes before flow cytometry detection, and the flow detection of the beta-galactosidase inside and outside the cells is innovatively realized by adopting a labeling method of a beta-galactosidase primary antibody combined with a fluorescence labeled secondary antibody, so that the synchronous labeling and detection of the protein inside and outside the lymphocytes are realized, and the detection efficiency of the immune cell beta-galactosidase is improved. Furthermore, the flow detection of the intracellular beta-galactosidase is combined with the detection of the surface marker of the conventional aged immune cell, so that the efficiency and the accuracy of immune cell detection are improved. For example, the step of washing the peripheral blood lymphocytes with a cell breaker is preceded by staining the peripheral blood lymphocytes with a flow antibody CD3-FITC, whereby the accuracy of T cell detection in the peripheral blood lymphocytes can be improved. Alternatively, peripheral blood lymphocytes were stained with flow-through antibodies CD3-FITC and CD56-PC5.5, so that the accuracy of NK cell detection in peripheral blood lymphocytes could be improved. Alternatively, the peripheral blood lymphocytes are stained with flow antibodies CD3-FITC and CD19-PC5.5, so that the accuracy of B cell detection in peripheral blood lymphocytes can be improved.
Specifically, the peripheral blood lymphocyte test sample includes at least one of T lymphocytes, B lymphocytes and NK cells. And obtaining the proportion of the aged lymphocytes in the peripheral blood lymphocyte to-be-detected sample according to the detection result of the flow cytometer. By combining the flow detection of the intracellular beta-galactosidase with the detection of the surface marker of the conventional aging immune cell for comparison, the aging conditions of T cells, NK cells and B cells in human peripheral blood can be detected more comprehensively, and the accuracy of the aging detection of the immune cell is improved.
In order to verify the accuracy of the detection method, the sample is subjected to four experiments, namely total lymphocyte detection, T cell detection, NK cell detection and B cell detection. Each test experiment is divided into two groups, one group is used as a negative pairIn the control (NC group), the other group was treated with low-concentration hydrogen peroxide (50. mu. mol/L) for 1 hour to induce senescence (H)2O2Groups), three replicates were set up for each group. Finally, the application provides a more convenient, sensitive and accurate quantitative detection method for detecting beta-galactosidase of immune cells.
The following description will be given with reference to specific examples.
Experimental Total lymphocyte detection
1.1 isolation of human peripheral blood lymphocytes
50mL of peripheral blood is treated by using a human peripheral blood lymphocyte separation solution to separate total lymphocytes, and the steps are as follows:
1) centrifuging fresh heparin anticoagulated volunteer peripheral blood 50mL, 700g for 20 min;
2) taking the upper plasma layer, carrying out water bath at 56 ℃ for 30min, keeping the temperature at 4 ℃ for 15min, finally centrifuging 2000g for 15min, taking the supernatant inactivated plasma to a new tube, and storing at 4 ℃ for later use;
3) adding DPBS into the lower layer of the centrifuged blood to the original volume of blood, and slowly adding the DPBS onto the liquid level of the lymphocyte separation along the tube wall;
4) centrifuging at 800g for 15 min;
5) collecting the centrifuged annular milky white lymphocytes, adding the collected annular milky white lymphocytes into a 50mL centrifuge tube, supplementing DPBS to 50mL, centrifuging for 10min at 600g, discarding the supernatant, resuspending the collected annular milky white lymphocytes by 50mL DPBS, and sampling and counting.
1.2 treatment of human peripheral blood lymphocyte samples before flow cytometry
Total lymphocyte assay fractions were divided into two groups, one group was used as a negative control (NC group), and the other group was treated with low-concentration hydrogen peroxide (50. mu. mol/L) for 1H to induce senescence (H)2O2Groups), three replicates were set up for each group and treated as follows.
The two groups are respectively 2 × 106Individual peripheral blood lymphocytes, each group was divided into two equal portions and treated as follows: comparing the scheme: the cells were collected by centrifugation into 1.5ml centrifuge tubes, washed 1 time with DPBS, and stained with 1 ml of beta-galactosidaseFixing the liquid (Biyun day, C0602) at room temperature for 15 min; centrifuging, removing the cell fixative by aspiration, and washing the cells with DPBS for 3 times, each for 3 minutes; removing DPBS by suction, adding 0.5 ml of staining working solution into each tube, and incubating overnight at 37 ℃; and (3) taking part of the stained cells, dripping the stained cells onto a glass slide or into a 6-well plate, and observing and photographing under a common optical microscope. The scheme of the application: after 1 time of DPBS washing, washing with intracellular staining and penetrating washing liquid (membrane breaking agent) for 1 time, then resuspending, adding beta-galactosidase (beta-GAL) rabbit primary antibody (Abcam, ab220283), standing at 37 ℃ for 1h, adding PE labeled mouse anti-rabbit IgG secondary antibody (southern Biotech, 4090-09), incubating for 15-20 min at room temperature in a dark place, after 1 time of each washing with intracellular staining and penetrating washing liquid and DPBS, resuspending cells with DPBS, and detecting lymphocytes with an up-flow cytometer.
1.3 analysis of results:
two groups of total lymphocyte senescence assays are shown in FIG. 1, and the statistical analysis is if: NC group and H2O2The proportion of the blue-stained senescent lymphocytes is respectively (5.00 +/-1.00)%, and (5.33 +/-1.53)%, and the difference has no statistical significance; NC group and H2O2beta-GAL for detecting senescence by flow cytometry+The lymphocyte ratios were (14.49. + -. 0.55)%, and (25.93. + -. 2.08)%, respectively, and the differences were statistically significant (FIG. 2).
Experimental two T cell detection
2.1 human peripheral blood lymphocyte isolation
Same as in experiment one.
2.2 treatment of human peripheral blood lymphocyte samples before flow cytometry
The T cell assay was divided into two groups, one of which was used as a negative control (NC group) and the other of which was treated with low-concentration hydrogen peroxide (50. mu. mol/L) for 1 hour to induce senescence (H)2O2Groups), three replicates were set up for each group and treated as follows.
The two groups are respectively 2 × 106Individual peripheral blood lymphocytes, each group was divided into two equal portions and treated as follows: comparing the scheme: staining with flow-through antibodies CD3-FITC, CD57-PE according to the antibody instructions; the scheme of the application: according to the resistanceAfter staining with a flow antibody CD3-FITC according to an instruction for body use, fixing with 4% paraformaldehyde (a fixative) at room temperature, washing with DPBS for 1 time, washing with an intracellular staining infiltration washing solution (a membrane breaking agent) for 1 time, then resuspending, adding a beta-galactosidase (beta-GAL) rabbit-derived primary antibody (Abcam, ab220283), standing at 37 ℃ for 1h, adding a PE-labeled mouse anti-rabbit IgG secondary antibody (southern Biotech, 4090-09), incubating at room temperature in a dark place for 15-20 min, washing with the intracellular staining infiltration washing solution and DPBS for 1 time respectively, and then resuspending the cells with the DPBS. The samples are detected by a flow cytometer.
2.3 analysis of results
Flow assay of the aging ratio of T cells in both groups is shown in fig. 3, and statistical analysis is given if: NC group and H2O2Group aged CD3+CD57+The proportion of T cells is (10.57 +/-1.74)%, and (22.65 +/-2.41)%, respectively, and the difference has statistical significance; NC group and H2O2Group aged CD3+β-GAL+The T cell ratios were (16.76. + -. 1.55)%, and (30.52. + -. 1.59)%, respectively, and the differences were statistically significant (FIG. 4).
Experimental three NK cell detection
3.1 human peripheral blood lymphocyte isolation
Same as in experiment one.
3.2 treatment of human peripheral blood lymphocyte samples before flow cytometry
NK cell assay was divided into two groups, one group was used as a negative control (NC group), and the other group was treated with low-concentration hydrogen peroxide (50. mu. mol/L) for 1H to induce senescence (H)2O2Groups), three replicates were set up for each group and treated as follows.
The two groups are respectively 2 × 106Individual peripheral blood lymphocytes, each group was divided into two equal portions and treated as follows: comparing the scheme: staining with flow-through antibodies CD3-FITC, CD56-PC5.5 and CD57-PE according to the antibody instructions; the scheme of the application: after staining with flow-through antibodies CD3-FITC and CD56-PC5.5 according to the antibody instructions, fixation was performed at room temperature with 4% paraformaldehyde (fixative), and after 1 DPBS washing, the cells were washed with intracellular staining and penetrating wash solution (membrane breaker)After 1 time, resuspension is carried out, beta-galactosidase rabbit primary antibody (Abcam, ab220283) is added, after the mixture is placed for 1 hour at 37 ℃, PE-labeled mouse anti-rabbit IgG secondary antibody (southern Biotech, 4090-09) is added, after incubation for 15-20 min at room temperature in a dark place, cells are resuspended by using intracellular staining and permeation washing liquid and DPBS (DPBS) for 1 time respectively. The samples are detected by a flow cytometer.
3.3 analysis of results
Two sets of flow assays for the proportion of NK cell senescence are shown in FIG. 5, and statistical analysis is given if: NC group and H2O2Group aged CD3-CD56+CD57+The NK cell proportion is respectively (12.66 +/-2.02)%, (30.68 +/-2.36)%, and the difference has statistical significance; NC group and H2O2Group aged CD3-CD56+β-GAL+The NK cell ratios were (16.96. + -. 1.98)%, and (33.96. + -. 1.24)%, respectively, and the differences were statistically significant (FIG. 6).
Experimental four B cell assay
4.1 human peripheral blood lymphocyte isolation
Same as in experiment one.
4.2 treatment of human peripheral blood lymphocyte samples before flow cytometry
The B cell assay was divided into two groups, one of which was used as a negative control (NC group) and the other was treated with low-concentration hydrogen peroxide (50. mu. mol/L) for 1H to induce senescence (H)2O2Groups), three replicates were set up for each group and treated as follows.
The two groups are respectively 2 × 106Individual peripheral blood lymphocytes, each group was divided into two equal portions and treated as follows: comparing the scheme: staining with flow-through antibodies CD3-FITC, CD19-PC5.5 and IgD-PE according to the antibody instructions; the embodiment group: after staining with flow-through antibodies CD3-FITC and CD19-PC5.5 according to the antibody instructions, fixation was performed at room temperature with 4% paraformaldehyde (fixative), after 1 time of DPBS washing, washing was performed with intracellular staining penetration wash (membrane breaker) 1 time and then resuspended, and after adding beta-galactosidase rabbit primary antibody (Abcam, ab220283), after standing at 37 ℃ for 1h, PE-labeled mouse anti-rabbit IgG secondary antibody (southern Biotech, 4090) was addedAnd-09), incubating for 15-20 min at room temperature in a dark place, washing the cells for 1 time respectively by using an intracellular staining and permeating washing solution and DPBS, and then resuspending the cells by the DPBS. The samples are detected by a flow cytometer.
4.3 analysis of results
Flow assays of the B cell senescence ratios in both groups are shown in FIG. 7, and statistical analysis was performed if: NC group and H2O2Group aged CD3-CD19+IgD-The proportion of B cells is (5.92 +/-0.92)%, and (17.28 +/-1.18)%, respectively, and the difference has statistical significance; NC group and H2O2Group aged CD3-CD19+β-GAL+The proportion of B cells was (9.04. + -. 0.48)%, respectively, (20.04. + -. 1.77)%, and the difference was statistically significant (FIG. 8).
In summary, the results of the total lymphocyte assay in experiment one show that: detection of NC group and H by traditional beta-galactosidase staining method2O2The proportion of the aged lymphocytes in the group is not obviously different, but the beta-GAL flow detection result is obviously different, so that the detection method has higher accuracy compared with the traditional detection method. In experiments II, III and IV, flow detection results of various immune cells by fluorescent antibody labeled cell surface and intracellular senescence-associated proteins show that: h2O2The proportion of the aged immune cells of the group is obviously higher than that of a negative control group (NC group), so that the detection method has good sensitivity; and the proportion of beta-GAL positive cells in each group is obviously higher than that of the traditional immune cell senescence marker protein positive cells, so that the application proves that the flow detection of the senescence marker protein beta-GAL in the immune cells can more accurately detect the senescent immune cells.
The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (10)
1. A sample pretreatment method for flow cytometry is characterized by comprising the following steps:
extracting peripheral blood lymphocytes;
and washing the peripheral blood lymphocytes by using a membrane breaking agent, and adding a beta-galactosidase primary antibody and a fluorescence-labeled secondary antibody for incubation treatment.
2. The method for pretreating a sample according to claim 1, wherein the step of adding a primary β -galactosidase antibody and a fluorescently-labeled secondary antibody for incubation comprises: and (3) firstly adding the beta-galactosidase primary antibody, standing for 40-80min, and then adding the fluorescence-labeled secondary antibody, and incubating for 15-20 min in a dark place.
3. The method for pretreating a sample according to claim 1, wherein the step of adding the primary β -galactosidase antibody and the fluorescently-labeled secondary antibody for incubation further comprises washing with the membrane-breaking agent and the buffer solution in sequence.
4. The method for pretreating a specimen according to claim 1, wherein the β -galactosidase primary antibody is a β -galactosidase rabbit-derived primary antibody; and/or the presence of a gas in the gas,
the fluorescence-labeled secondary antibody is a phycoerythrin-labeled mouse anti-rabbit immunoglobulin secondary antibody.
5. The method for pretreating a sample according to claim 1, wherein the disrupting agent is a Triton X-100 solution.
6. The method for pretreating a sample according to any one of claims 1 to 5, wherein said step of washing said peripheral blood lymphocytes with a cell breaker is preceded by the step of:
staining the peripheral blood lymphocytes with the flow antibody CD3-FITC, or staining the peripheral blood lymphocytes with the flow antibodies CD3-FITC and CD56-PC5.5, or staining the peripheral blood lymphocytes with the flow antibodies CD3-FITC and CD19-PC 5.5; then, fixing treatment is carried out by using a fixing agent.
7. The method for pretreating a specimen according to claim 6, wherein the fixing agent is a paraformaldehyde fixing solution.
8. An immune cell detection method, characterized by comprising the steps of;
providing a peripheral blood lymphocyte sample to be tested; the peripheral blood lymphocyte test sample is a sample treated by the sample pretreatment method according to any one of claims 1 to 7;
and detecting the peripheral blood lymphocyte sample to be detected by using a flow cytometer.
9. The method according to claim 8, wherein the test sample of peripheral blood lymphocytes comprises at least one of T lymphocytes, B lymphocytes and NK cells.
10. The method for detecting an immune cell according to claim 8, wherein the proportion of senescent lymphocytes in the sample for peripheral blood lymphocyte measurement is obtained based on the result of the detection by the flow cytometer.
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| CN113804610A (en) * | 2021-09-22 | 2021-12-17 | 深圳中检联新药检测有限责任公司 | Detection method for mesenchymal stem cell aging |
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Application publication date: 20210611 |