CN113029733B - Living cell immunofluorescence staining method based on labeled antibody - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000003125 immunofluorescent labeling Methods 0.000 title claims abstract description 23
- 239000011259 mixed solution Substances 0.000 claims abstract description 99
- 239000000243 solution Substances 0.000 claims abstract description 50
- 238000002156 mixing Methods 0.000 claims abstract description 33
- 238000010166 immunofluorescence Methods 0.000 claims abstract description 29
- 239000012752 auxiliary agent Substances 0.000 claims abstract description 27
- 239000006143 cell culture medium Substances 0.000 claims abstract description 22
- 238000004043 dyeing Methods 0.000 claims abstract description 21
- 238000012258 culturing Methods 0.000 claims abstract description 11
- 238000005406 washing Methods 0.000 claims abstract description 11
- 229920002873 Polyethylenimine Polymers 0.000 claims description 16
- 238000001816 cooling Methods 0.000 claims description 16
- 239000008367 deionised water Substances 0.000 claims description 16
- 229910021641 deionized water Inorganic materials 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 8
- 230000003834 intracellular effect Effects 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 102000007999 Nuclear Proteins Human genes 0.000 claims description 4
- 108010089610 Nuclear Proteins Proteins 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000002372 labelling Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 82
- 210000000170 cell membrane Anatomy 0.000 abstract description 9
- 238000012757 fluorescence staining Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 description 16
- 210000004698 lymphocyte Anatomy 0.000 description 15
- 238000001514 detection method Methods 0.000 description 11
- 238000010186 staining Methods 0.000 description 10
- 239000007850 fluorescent dye Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- 101710118538 Protease Proteins 0.000 description 3
- 230000030833 cell death Effects 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000010212 intracellular staining Methods 0.000 description 3
- 238000007447 staining method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 241000283707 Capra Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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Abstract
The application discloses a living cell immunofluorescence staining method based on a labeled antibody, which comprises the following steps: a1: fully mixing the live cell immunofluorescence auxiliary agent and opt-MEM culture solution according to the volume part ratio of 1:12.5, and incubating for 15min at room temperature to obtain a mixed solution 1; a2: fully mixing the living cell antibody and the opt-MEM culture solution according to the volume part ratio of 1:50, and incubating for 15min at room temperature to obtain a mixed solution 2; a3: fully mixing the mixed solution 1 and the mixed solution 2 according to the volume part ratio of 54:52, and incubating for 15min at room temperature to obtain a mixed solution 3; a4: adding the mixed solution 3 into a target cell culture medium to obtain a mixed solution 4, wherein the volume part ratio of the mixed solution 3 to the target cell culture medium is 53:100; a5: culturing the mixed solution 4 for 4-6 hours, and washing with PBS solution for 2-3 times to finish dyeing. According to the application, cell membrane perforation is not needed, and the antibody is utilized, so that the fluorescence staining range of living cells is the same as the immunofluorescence staining range of fixed cells; meanwhile, the method only needs 4-6 hours, and the time is obviously shortened.
Description
Technical Field
The application relates to the application fields of immunoassay and biotechnology, in particular to a living cell immunofluorescence staining method based on a labeled antibody.
Background
The current domestic living cell staining method comprises the following steps: 1. chinese patent "immunofluorescence labeling method of living intracellular proteins", patent number "201410061287.9", which uses liposomes as carriers for intracellular staining, requires 22-26 hours and is directed only against antibodies directly bearing fluorescent labels; 2. chinese patent ' an immunofluorescence labeling method of protein in living cells ', patent No. 201910633999.6 ', has a certain influence on cell activity and has low staining efficiency.
Currently, staining of living cells is generally achieved by chemical fluorochromes, which have a very limited range of staining and are not protein-specific but chemical structure-specific. At present, conventional fluorescent staining is generally realized through cell membrane perforation, the cell activity is influenced by the cell membrane perforation, cell death is easy to cause, the generation of cell fragments is increased, and the flow cytometry detection result is influenced. At present, the conventional fluorescent staining time is longer, and the experimental efficiency is affected.
Disclosure of Invention
Aiming at the problems existing in the prior art, the application provides a living cell immunofluorescence staining method based on a labeled antibody, which does not need to punch cell membranes, and uses the antibody to ensure that the fluorescence staining range of living cells is the same as the fixed cell immunofluorescence staining range; meanwhile, the method only needs 4-6 hours, and the time is obviously shortened.
The application is realized by the following technical scheme: a method of immunofluorescent staining of living cells based on labeled antibodies, comprising the steps of:
a1: fully mixing the live cell immunofluorescence auxiliary agent and opt-MEM culture solution according to the volume part ratio of 0.5-1.5:12-13, and incubating for 10-15min at room temperature to obtain a mixed solution 1;
a2: fully mixing the living cell antibody and opt-MEM culture solution according to the volume part ratio of 0.5-1.5:45-55, and incubating for 10-15min at room temperature to obtain a mixed solution 2;
a3: fully mixing the mixed solution 1 and the mixed solution 2 according to the volume part ratio of 50-55:50-55, and incubating for 10-15min at room temperature to obtain a mixed solution 3;
a4: adding the mixed solution 3 into a target cell culture medium to obtain a mixed solution 4, wherein the volume part ratio of the mixed solution 3 to the target cell culture medium is 50-55:100;
a5: culturing the mixed solution 4 for 4-6 hours, and washing with PBS solution for 2-3 times to finish dyeing.
Further, the method comprises the following steps:
a1: fully mixing the live cell immunofluorescence auxiliary agent and opt-MEM culture solution according to the volume part ratio of 1:12.5, and incubating for 15min at room temperature to obtain a mixed solution 1;
a2: fully mixing the living cell antibody and the opt-MEM culture solution according to the volume part ratio of 1:50, and incubating for 15min at room temperature to obtain a mixed solution 2;
a3: fully mixing the mixed solution 1 and the mixed solution 2 according to the volume part ratio of 54:52, and incubating for 15min at room temperature to obtain a mixed solution 3;
a4: adding the mixed solution 3 into a target cell culture medium to obtain a mixed solution 4, wherein the volume part ratio of the mixed solution 3 to the target cell culture medium is 53:100;
a5: culturing the mixed solution 4 for 4-6 hours, and washing with PBS solution for 2-3 times to finish dyeing.
Further, in the step A2, the living cell antibody is a mixture of primary and secondary antibodies of nuclear endoprotease, and the weight ratio of the primary and secondary antibodies is 1:1.
Further, in the step A2, the living cell antibody is a fluorescent-labeled intracellular protein antibody.
Further, in the step A1, the preparation method of the living cell immunofluorescence auxiliary agent comprises the following steps:
b1: heating deionized water to 70-90 ℃, adding linear polyethylenimine, fully dissolving, and cooling to room temperature to obtain a cooling solution, wherein the weight parts ratio of the deionized water to the linear polyethylenimine is as follows: 500:0.5-1.5;
b2: the pH of the cooled solution was adjusted to 6-8, and then filtered through a 0.22 μm syringe filter, and the temperature was kept at 3-6deg.C after filtration to obtain a live cell immunofluorescence auxiliary agent.
Further, in the step A1, the preparation method of the living cell immunofluorescence auxiliary agent comprises the following steps:
b1: heating deionized water to 80 ℃, adding linear polyethylenimine, fully dissolving, and cooling to room temperature to obtain a cooling solution, wherein the weight parts ratio of the deionized water to the linear polyethylenimine is as follows: 500:1;
b2: the pH of the cooled solution was adjusted to 7, and then filtered with a 0.22 μm syringe filter, and the temperature was kept at 4℃after the filtration, to obtain a live cell immunofluorescence auxiliary agent.
The above-mentioned at least includes following advantage:
1. according to the living cell immunofluorescence staining method based on the labeled antibody, cell membrane perforation is not needed, and the antibody is utilized, so that the fluorescence staining range of living cells is the same as the fixed cell immunofluorescence staining range;
2. the living cell immunofluorescence staining method based on the labeled antibody can perform deflection staining in 4-6 hours, and the time is obviously shortened;
3. the living cell immunofluorescence staining method based on the labeled antibody can expand the fluorescence staining range, and has more specificity than the existing chemical fluorescent dye.
Drawings
The accompanying drawings, which are included to provide a further understanding of embodiments of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:
FIG. 1 is a graph showing the effect of an indirectly labeled antibody of the present application on staining living cells under a fluorescence microscope;
FIG. 2 is a graph showing the comparison of the staining effect of an indirectly labeled antibody of the present application on living cells with the intracellular staining effect of living cells of the prior art;
FIG. 3 is a graph showing the effect of the direct labeled antibodies of the present application on staining living cells under a fluorescence microscope;
FIG. 4 is a graph showing the comparison of the staining effect of the direct labeled antibody of the present application on living cells with the intracellular staining effect of living cells of the prior art.
Detailed Description
For the purpose of making apparent the objects, technical solutions and advantages of the present application, the present application will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present application and the descriptions thereof are for illustrating the present application only and are not to be construed as limiting the present application.
The application relates to a living cell immunofluorescence staining method based on a labeled antibody, which comprises the following steps:
a1: fully mixing the live cell immunofluorescence auxiliary agent and opt-MEM culture solution according to the volume part ratio of 0.5-1.5:12-13, and incubating for 10-15min at room temperature to obtain a mixed solution 1;
a2: fully mixing the living cell antibody and opt-MEM culture solution according to the volume part ratio of 0.5-1.5:45-55, and incubating for 10-15min at room temperature to obtain a mixed solution 2;
a3: fully mixing the mixed solution 1 and the mixed solution 2 according to the volume part ratio of 50-55:50-55, and incubating for 10-15min at room temperature to obtain a mixed solution 3;
a4: adding the mixed solution 3 into a target cell culture medium to obtain a mixed solution 4, wherein the volume part ratio of the mixed solution 3 to the target cell culture medium is 50-55:100;
a5: culturing the mixed solution 4 for 4-6 hours, and washing with PBS solution for 2-3 times to finish dyeing.
More preferably, the method comprises the steps of:
a1: fully mixing the live cell immunofluorescence auxiliary agent and opt-MEM culture solution according to the volume part ratio of 0.5:12, and incubating for 15min at room temperature to obtain a mixed solution 1;
a2: fully mixing the living cell antibody and the opt-MEM culture solution according to the volume part ratio of 0.5:45, and incubating for 15min at room temperature to obtain a mixed solution 2;
a3: fully mixing the mixed solution 1 and the mixed solution 2 according to the volume part ratio of 1:1.1, and incubating for 15min at room temperature to obtain a mixed solution 3;
a4: adding the mixed solution 3 into a target cell culture medium to obtain a mixed solution 4, wherein the volume part ratio of the mixed solution 3 to the target cell culture medium is 50:100;
a5: culturing the mixed solution 4 for 4-6 hours, and washing with PBS solution for 2-3 times to finish dyeing.
More preferably, the method comprises the steps of:
a1: fully mixing the live cell immunofluorescence auxiliary agent and opt-MEM culture solution according to the volume part ratio of 1.5:13, and incubating for 15min at room temperature to obtain a mixed solution 1;
a2: fully mixing the living cell antibody and the opt-MEM culture solution according to the volume part ratio of 1.5:55, and incubating for 15min at room temperature to obtain a mixed solution 2;
a3: fully mixing the mixed solution 1 and the mixed solution 2 according to the volume part ratio of 1:1, and incubating for 15min at room temperature to obtain a mixed solution 3;
a4: adding the mixed solution 3 into a target cell culture medium to obtain a mixed solution 4, wherein the volume part ratio of the mixed solution 3 to the target cell culture medium is 55:100;
a5: culturing the mixed solution 4 for 4-6 hours, and washing with PBS solution for 2-3 times to finish dyeing.
More preferably, the method comprises the steps of:
a1: fully mixing the live cell immunofluorescence auxiliary agent and opt-MEM culture solution according to the volume part ratio of 1:12.5, and incubating for 15min at room temperature to obtain a mixed solution 1;
a2: fully mixing the living cell antibody and the opt-MEM culture solution according to the volume part ratio of 1:50, and incubating for 15min at room temperature to obtain a mixed solution 2;
a3: fully mixing the mixed solution 1 and the mixed solution 2 according to the volume part ratio of 54:52, and incubating for 15min at room temperature to obtain a mixed solution 3;
a4: adding the mixed solution 3 into a target cell culture medium to obtain a mixed solution 4, wherein the volume part ratio of the mixed solution 3 to the target cell culture medium is 53:100;
a5: culturing the mixed solution 4 for 4-6 hours, and washing with PBS solution for 2-3 times to finish dyeing.
More preferably, in the step A2, the living cell antibody is a mixture of primary and secondary antibodies of nuclear endoprotease, and the weight ratio of the primary and secondary antibodies is 1:1.
More preferably, in the step A2, the living cell antibody is a fluorescent-labeled intracellular protein antibody.
More preferably, in the step A1, the preparation method of the living cell immunofluorescence auxiliary agent includes the following steps:
b1: heating deionized water to 70-90 ℃, adding linear polyethylenimine, fully dissolving, and cooling to room temperature to obtain a cooling solution, wherein the weight parts ratio of the deionized water to the linear polyethylenimine is as follows: 500:0.5-1.5;
b2: the pH of the cooled solution was adjusted to 6-8, and then filtered through a 0.22 μm syringe filter, and the temperature was kept at 3-6deg.C after filtration to obtain a live cell immunofluorescence auxiliary agent.
More preferably, in the step A1, the preparation method of the living cell immunofluorescence auxiliary agent includes the following steps:
b1: heating deionized water to 80 ℃, adding linear polyethylenimine, fully dissolving, and cooling to room temperature to obtain a cooling solution, wherein the weight parts ratio of the deionized water to the linear polyethylenimine is as follows: 500:1;
b2: the pH of the cooled solution was adjusted to 7, and then filtered with a 0.22 μm syringe filter, and the temperature was kept at 4℃after the filtration, to obtain a live cell immunofluorescence auxiliary agent.
The preferred embodiments of the process method of the present application are as follows.
Example 1
A method of immunofluorescent staining of living cells based on labeled antibodies, comprising the steps of:
a1: fully mixing the live cell immunofluorescence auxiliary agent and opt-MEM culture solution according to the volume part ratio of 1:12.5, and incubating for 15min at room temperature to obtain a mixed solution 1;
a2: fully mixing the living cell antibody and the opt-MEM culture solution according to the volume part ratio of 1:50, and incubating for 15min at room temperature to obtain a mixed solution 2;
a3: fully mixing the mixed solution 1 and the mixed solution 2 according to the volume part ratio of 54:52, and incubating for 15min at room temperature to obtain a mixed solution 3;
a4: adding the mixed solution 3 into a target cell culture medium to obtain a mixed solution 4, wherein the volume part ratio of the mixed solution 3 to the target cell culture medium is 53:100;
a5: culturing the mixed solution 4 for 4-6 hours, and washing with PBS solution for 2-3 times to finish dyeing.
More specifically, in the step A2, the living cell antibody is a mixture of primary and secondary antibodies of nuclear endoprotease, and the weight ratio of the primary and secondary antibodies is 1:1.
More specifically, the primary nuclear protein antibody is a rabbit lymphocyte nuclear protein antibody, and the secondary nuclear protein antibody is a goat anti-rabbit antibody with AF 488.
More specifically, in the step A4, the target cell is a mouse lymphocyte.
More specifically, in the step A1, the preparation method of the living cell immunofluorescence auxiliary agent comprises the following steps:
b1: heating deionized water to 80 ℃, adding linear polyethylenimine, fully dissolving, and cooling to room temperature to obtain a cooling solution, wherein the weight parts ratio of the deionized water to the linear polyethylenimine is as follows: 500:1;
b2: the pH of the cooled solution was adjusted to 7, and then filtered with a 0.22 μm syringe filter, and the temperature was kept at 4℃after the filtration, to obtain a live cell immunofluorescence auxiliary agent.
Example 2
A method of immunofluorescent staining of living cells based on labeled antibodies, comprising the steps of:
a1: fully mixing the live cell immunofluorescence auxiliary agent and opt-MEM culture solution according to the volume part ratio of 1:12.5, and incubating for 15min at room temperature to obtain a mixed solution 1;
a2: fully mixing the living cell antibody and the opt-MEM culture solution according to the volume part ratio of 1:50, and incubating for 15min at room temperature to obtain a mixed solution 2;
a3: fully mixing the mixed solution 1 and the mixed solution 2 according to the volume part ratio of 54:52, and incubating for 15min at room temperature to obtain a mixed solution 3;
a4: adding the mixed solution 3 into a target cell culture medium to obtain a mixed solution 4, wherein the volume part ratio of the mixed solution 3 to the target cell culture medium is 53:100;
a5: culturing the mixed solution 4 for 4-6 hours, and washing with PBS solution for 2-3 times to finish dyeing.
More specifically, in the step A2, the living cell antibody is a fluorescent-labeled intracellular protein antibody.
More specifically, the intracellular protein antibody is a lymphocyte-secreted protein antibody, specifically an ifnγ antibody with an APC fluorescent label.
More specifically, in the step A4, the target cell is a mouse lymphocyte.
More specifically, in the step A1, the preparation method of the living cell immunofluorescence auxiliary agent comprises the following steps:
b1: heating deionized water to 80 ℃, adding linear polyethylenimine, fully dissolving, and cooling to room temperature to obtain a cooling solution, wherein the weight parts ratio of the deionized water to the linear polyethylenimine is as follows: 500:1;
b2: the pH of the cooled solution was adjusted to 7, and then filtered with a 0.22 μm syringe filter, and the temperature was kept at 4℃after the filtration, to obtain a live cell immunofluorescence auxiliary agent.
Experimental example 1
The stained mouse lymphocytes of example 1 were observed. As shown in fig. 1, fig. 1 (a) is a view of mouse lymphocytes observed under a normal microscope at 400 x, and the mouse lymphocytes can be clearly observed; FIG. 1 (b) is a diagram of labeled mouse lymphocytes observed under an immunofluorescence microscope at 400 Xmagnification, and stained markers in the nuclei of mouse lymphocytes are observed; FIG. 1 (c) is a graph of FIG. 1 (a) and FIG. 1 (b) combined to one, and the proportion of stained marker cells to total cell number in the nuclei of the marked mouse lymphocytes can be observed. As can be seen from FIG. 1, immunofluorescence staining can be achieved without perforating by using the living cell immunofluorescence staining method of the present application.
As shown in fig. 2, fig. 2 (a) is a graph showing the effect of the prior art staining method on the nuclei, followed by detection by a flow cytometer; FIG. 2 (b) is a graph showing the effect of the indirectly labeled antibody of the present application on staining living cells, followed by detection by a flow cytometer. As can be seen from the graph, the detection effect of the application is equivalent to that of the prior art, and no obvious difference exists. However, compared with the prior art, the dyeing method of the application needs 22-26 hours, and the dyeing method of the application only needs 4-6 hours; in the prior art, cell membrane perforation is needed during dyeing, cell activity is affected by cell membrane perforation, cell death is easy to cause, generation of cell fragments is increased, flow cytometry detection results are affected, a large failure rate exists, multiple groups of experiments are needed to obtain accurate results, and the application does not need perforation and does not affect detection results; as can be seen from FIG. 2, the dyeing method of the present application can expand the range of fluorescent dyeing, and has more specificity than the existing chemical fluorescent dyes.
Experimental example 2
The stained mouse lymphocytes of example 2 were observed. As shown in fig. 3, fig. 3 (a) is a view of mouse lymphocytes observed under a normal microscope at 400 x, and the mouse lymphocytes can be clearly observed; FIG. 3 (b) is a photograph of labeled mouse lymphocytes observed under an immunofluorescence microscope at 400 Xmagnification, and stained labeling of the mouse lymphocytes in cells can be observed; FIG. 3 (c) is a graph of FIG. 3 (a) and FIG. 3 (b) combined to one, and the proportion of stained marker cells to total cell number in the cells of the marked mouse lymphocytes can be observed. As can be seen from FIG. 3, immunofluorescence staining can be achieved without perforating by using the living cell immunofluorescence staining method of the present application.
As shown in fig. 4, fig. 4 (a) is a graph showing the effect of the prior art staining method on intracellular cells, followed by detection by a flow cytometer; FIG. 4 (b) is a graph showing the effect of the indirectly labeled antibody of the present application on staining living cells, followed by detection by a flow cytometer. As can be seen from the graph, the detection effect of the application is equivalent to that of the prior art, and no obvious difference exists. However, compared with the prior art, the dyeing method of the application needs 22-26 hours, and the dyeing method of the application only needs 4-6 hours; in the prior art, cell membrane perforation is needed during dyeing, cell activity is affected by cell membrane perforation, cell death is easy to cause, generation of cell fragments is increased, flow cytometry detection results are affected, a large failure rate exists, multiple groups of experiments are needed to obtain accurate results, and the application does not need perforation and does not affect detection results; as can be seen from FIG. 4, the dyeing method of the present application can expand the range of fluorescent dyeing, and has more specificity than the existing chemical fluorescent dyes.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the application, and is not meant to limit the scope of the application, but to limit the application to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the application are intended to be included within the scope of the application.
Claims (3)
1. A method of immunofluorescent staining living cells based on labeled antibodies, comprising the steps of:
a1: fully mixing the live cell immunofluorescence auxiliary agent and opt-MEM culture solution according to the volume part ratio of 0.5-1.5:12-13, and incubating for 10-15min at room temperature to obtain a mixed solution 1;
a2: fully mixing the living cell antibody and opt-MEM culture solution according to the volume part ratio of 0.5-1.5:45-55, and incubating for 10-15min at room temperature to obtain a mixed solution 2;
a3: fully mixing the mixed solution 1 and the mixed solution 2 according to the volume part ratio of 50-55:50-55, and incubating for 10-15min at room temperature to obtain a mixed solution 3;
a4: adding the mixed solution 3 into a target cell culture medium to obtain a mixed solution 4, wherein the volume part ratio of the mixed solution 3 to the target cell culture medium is 50-55:100;
a5: culturing the mixed solution 4 for 4-6 hours, and washing with PBS solution for 2-3 times to finish dyeing;
in the step A2, the living cell antibody is a mixture of primary and secondary antibodies of nuclear protein, and the weight part ratio of the primary antibody to the secondary antibody is 1:1; or in the step A2, the living cell antibody is a fluorescent-labeled intracellular protein antibody;
in the step A1, the preparation method of the living cell immunofluorescence auxiliary agent comprises the following steps:
b1: heating deionized water to 70-90 ℃, adding linear polyethylenimine, fully dissolving, and cooling to room temperature to obtain a cooling solution, wherein the weight parts ratio of the deionized water to the linear polyethylenimine is as follows: 500:0.5-1.5;
b2: the pH of the cooled solution was adjusted to 6-8, and then filtered through a 0.22 μm syringe filter, and the temperature was kept at 3-6deg.C after filtration to obtain a live cell immunofluorescence auxiliary agent.
2. The method of claim 1, comprising the steps of:
a1: fully mixing the live cell immunofluorescence auxiliary agent and opt-MEM culture solution according to the volume part ratio of 1:12.5, and incubating for 15min at room temperature to obtain a mixed solution 1;
a2: fully mixing the living cell antibody and the opt-MEM culture solution according to the volume part ratio of 1:50, and incubating for 15min at room temperature to obtain a mixed solution 2;
a3: fully mixing the mixed solution 1 and the mixed solution 2 according to the volume part ratio of 54:52, and incubating for 15min at room temperature to obtain a mixed solution 3;
a4: adding the mixed solution 3 into a target cell culture medium to obtain a mixed solution 4, wherein the volume part ratio of the mixed solution 3 to the target cell culture medium is 53:100;
a5: culturing the mixed solution 4 for 4-6 hours, and washing with PBS solution for 2-3 times to finish dyeing.
3. The method for labeling antibody-based living cell immunofluorescence staining according to claim 1, wherein in the step A1, the method for preparing the living cell immunofluorescence auxiliary agent comprises the steps of:
b1: heating deionized water to 80 ℃, adding linear polyethylenimine, fully dissolving, and cooling to room temperature to obtain a cooling solution, wherein the weight parts ratio of the deionized water to the linear polyethylenimine is as follows: 500:1;
b2: the pH of the cooled solution was adjusted to 7, and then filtered with a 0.22 μm syringe filter, and the temperature was kept at 4℃after the filtration, to obtain a live cell immunofluorescence auxiliary agent.
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| CN101329230A (en) * | 2008-07-14 | 2008-12-24 | 中国人民解放军第三军医大学 | Improved method for dyeing immunofluorescence cell |
| CN106940266A (en) * | 2017-03-21 | 2017-07-11 | 上海美吉医学检验有限公司 | A kind of dyeing enhancing liquid and colouring method dyed for cell surface |
| CA3018691A1 (en) * | 2016-04-06 | 2017-10-12 | Alteogen, Inc. | Antibody-drug conjugates comprising antibodies modified with metal ion-binding motifs |
| CN109342153A (en) * | 2018-10-15 | 2019-02-15 | 卢克焕 | A kind of immunofluorescence dyeing method of buffalo stem spermatogonium like cell |
| CN111175112A (en) * | 2020-01-13 | 2020-05-19 | 浙江卫未生物医药科技有限公司 | Improved microcarrier living cell fluorescent staining method |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101329230A (en) * | 2008-07-14 | 2008-12-24 | 中国人民解放军第三军医大学 | Improved method for dyeing immunofluorescence cell |
| CA3018691A1 (en) * | 2016-04-06 | 2017-10-12 | Alteogen, Inc. | Antibody-drug conjugates comprising antibodies modified with metal ion-binding motifs |
| CN106940266A (en) * | 2017-03-21 | 2017-07-11 | 上海美吉医学检验有限公司 | A kind of dyeing enhancing liquid and colouring method dyed for cell surface |
| CN109342153A (en) * | 2018-10-15 | 2019-02-15 | 卢克焕 | A kind of immunofluorescence dyeing method of buffalo stem spermatogonium like cell |
| CN111175112A (en) * | 2020-01-13 | 2020-05-19 | 浙江卫未生物医药科技有限公司 | Improved microcarrier living cell fluorescent staining method |
Non-Patent Citations (1)
| Title |
|---|
| Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!;Nina Bono et al.;《pharmaceutics》;第12卷(第2期);第5-11页 * |
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