CN114317438A - Cell sample preparation method for immunofluorescence experiment - Google Patents
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- CN114317438A CN114317438A CN202111675599.5A CN202111675599A CN114317438A CN 114317438 A CN114317438 A CN 114317438A CN 202111675599 A CN202111675599 A CN 202111675599A CN 114317438 A CN114317438 A CN 114317438A
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- 210000004027 cell Anatomy 0.000 claims description 27
- 210000001178 neural stem cell Anatomy 0.000 claims description 19
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Abstract
The invention discloses a cell sample preparation method for an immunofluorescence experiment, and belongs to the field of cell sample preparation. The method comprises the following steps: 1) taking a cell culture, and replacing a culture medium in the cell culture with PBS; 2) centrifuging the cells onto a slide using a centrifuge funnel; 3) air drying for 5-15 min. The method of the invention can ensure that the integrity of the cell sample is better and the cell sample is attached to the glass slide more tightly, thereby improving the effect of the immunofluorescence experiment.
Description
Technical Field
The invention belongs to the field of cell sample preparation.
Background
The immunofluorescence assay is a technique for directly or indirectly labeling a target protein with an antibody that labels a fluorescent molecule (e.g., fluorescein that can be excited such as isothiocyanate (FITC), tetraethylrhodamine (RB200), tetramethylrhodamine isothiocyanate (TRITC), or a substance that generates fluorescence after being catalyzed by an enzyme such as 4-methylumbelliferone-beta-D galactoside). The direct method is to directly add a labeled specific fluorescent antibody on an antigen specimen to realize the detection of the antigen. The indirect method is that the primary antibody is firstly combined with the target protein, and then the secondary antibody marked by the fluorescent molecule is added to be combined with the primary antibody, so that the antigen can be detected.
In immunofluorescence experiments, the integrity of the cells and the ability to attach to slides are important to the success or failure of the experiment. Generally, a sample preparation method for immunofluorescence experiments includes the steps of: (1) coating the coverslip/cell slide with polylysine or Matrigel, etc.; (2) putting the coated cover glass/cell slide into a 24-hole plate (or a proper cell culture plate/dish), inoculating a proper amount of cells, and culturing in a carbon dioxide incubator for 4-6 h; (3) the medium was aspirated and gently washed 1 time with the appropriate amount of PBS. However, the above method has problems that the cell morphology is incomplete, the ability to attach the slide is not strong, and the fluorescent coloring effect is poor.
Centrifugation funnels, also known as cytofunnels, are devices that concentrate cells in a cell suspension (including interstitial fluid) onto a slide by centrifugation. When the cell enrichment funnel is used, the filter paper, the buckle and other parts are used for assisting the outlet at the bottom of the funnel to be tightly attached to a glass slide, then cell suspension is added into the funnel, centrifugation is carried out, cells can be attached to the glass slide, and enrichment of a sample is completed.
The centrifugal funnel is not used for sample preparation of immunofluorescence experiments at present.
Disclosure of Invention
The invention aims to solve the problems that: provides a preparation method of an immunofluorescence experiment sample, which ensures that the cell shape is complete, the attaching capacity is strong and the fluorescence coloring effect is good.
The technical scheme of the invention is as follows:
a cell sample preparation method for immunofluorescence experiments comprises the following steps:
1) taking a cell culture, and replacing a culture medium in the cell culture with a PBS buffer solution;
2) cells were enriched onto slides using a centrifuge funnel;
3) air drying for 5-15 min.
Further, the specific method of replacement in step 1) is to remove the upper layer culture medium after centrifugation, add PBS buffer, and resuspend.
Further, the centrifugation parameters are:
centrifuge at 100 Xg for 5 min.
Further, the centrifugation parameters of step 2) are:
centrifuge at 400 Xg for 5 min.
Further, the cell is a neural stem cell.
Further, the neural stem cell is a neural stem cell sphere.
Further, the neural stem cell is a rat neural stem cell sphere.
The invention also provides an application of the cell sample in detecting the protein in the neural stem cell; the cell sample is prepared according to the method;
the application steps are as follows:
1) taking a cell sample, adding acetone or 4% paraformaldehyde for fixation, washing with a PBS buffer solution, adding a transmembrane solution and a confining solution for incubation, removing the solution, washing with the PBS buffer solution, and adding primary antibody for incubation;
2) and (3) taking the incubated liquid, washing with PBS buffer solution, adding a fluorescence-labeled secondary antibody for incubation, washing with PBS, adding an anti-fluorescence quenching agent for incubation, and acquiring an image under a microscope.
Further, the protein comprises Nestin and Sox2 protein.
The PBS buffer solution used in the invention is the most widely used buffer solution in biochemical research, and the main component of the PBS buffer solution is Na2HPO4、KH2PO4NaCl and KCl. The configuration method comprises the following steps: weighing 8.0g NaCl, 0.2g KCl and 1.44g Na2HPO4、0.24g KH2PO4Dissolving in 800mL of distilled water, adjusting the solution to 7.4 with HCl, and adding distilled water to a constant volume of 1L to obtain 0.01M PBS buffer solution.
Compared with the traditional sample preparation method for immunofluorescence experiments, the method has the following beneficial effects:
the method of the invention ensures that the integrity of the cell sample is better, the cell sample is attached to the glass slide more tightly, and the fluorescence intensity is higher.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a drawing of a confocal laser scanning microscope
FIG. 2 is a photograph of a confocal laser scanning microscope (confocal laser microscopy) and a control group
Detailed Description
Example 1 comparison of the preparation method of rat neural stem cell sample for immunofluorescence assay of the present invention with conventional preparation method
This example detects Nestin, Sox2 protein from neural stem cell spheres.
1. Test group (method of the invention)
(1) Collecting neural stem cell balls into a centrifuge tube, and centrifuging for 5min at 100 Xg;
(2) removing the upper layer culture medium, adding PBS buffer solution, gently suspending, and centrifuging at 100 Xg for 5 min;
(3) removing the supernatant, adding PBS buffer solution, and gently resuspending to obtain suspension of the neural stem cell spheres;
(4) assembling the glass slide, the filter paper sheet and the centrifugal funnel, fitting the glass slide with the bottom outlet of the centrifugal funnel, adding 100 mu L of heavy suspension into the funnel, and centrifuging for 5min at 400 Xg; (Note: the centrifugal funnel is configured in the Cytospin4 centrifugal smear machine)
(5) Taking out the glass slide after the centrifugation is finished, and airing for 5-15 min;
(6) adding precooled acetone into the glass slide, fixing for 15min at 4 ℃ (or adding 4% paraformaldehyde at room temperature), and adding PBS to rinse the glass slide for 5 times; 0.5-1ml of membrane penetrating liquid and sealing liquid are dripped into each glass slide, and the glass slide is incubated for 60min at room temperature. Sucking off the liquid; washing with PBS for 5 times; 0.5ml of the primary antibody was diluted dropwise and incubated overnight at 4 ℃.
(7) PBS was washed 5 times, and diluted Secondary antibodies (Anti-rabbitIgG (H + L), F (ab')2 Fragment (Alexa Fluor 488Conjugate), Goat Anti-Mouse IgG (H + L) highlyCross-Adsorbed second Antibody, Alexa Fluor Plus 555) were added dropwise thereto in a total amount of 0.5ml, followed by incubation in the dark for 1 hour at room temperature. Washing with PBS for 5 times; absorbing excessive PBS by using absorbent paper, adding an anti-fluorescence quencher (containing DAPI), incubating for 15min in a dark place, sealing by using a cover glass, observing the result under a laser confocal microscope, adjusting the laser intensity, keeping the contrast between groups consistent, and collecting images.
2. Control group (Current conventional preparation method)
(1) Coating the cover glass with polylysine or Matrigel;
(2) placing the coated coverslip into 24-well plate (or suitable cell culture plate/dish), and inoculating to obtain cell density of 1 × 107Placing each/mL neural stem cell ball in a carbon dioxide incubator for culturing for 4-6 h;
(3) the medium was aspirated off and gently washed 1 time with PBS buffer;
(4) adding precooled acetone into the glass slide, fixing for 15min at 4 ℃ (or adding 4% paraformaldehyde at room temperature), and adding PBS to rinse the glass slide for 5 times; 0.5-1ml of membrane penetrating liquid and sealing liquid are dripped into each glass slide, and the glass slide is incubated for 60min at room temperature. Sucking off the liquid; washing with PBS for 5 times; 0.5ml of the primary antibody was diluted dropwise and incubated overnight at 4 ℃.
(5) PBS was washed 5 times, 0.5ml of diluted Secondary antibodies (Anti-rabbitIgG (H + L), F (ab')2 Fragment (Alexa Fluor 488Conjugate), Goat Anti-Mouse IgG (H + L) highlyCross-Adsorbed second Antibody, Alexa Fluor Plus 555) were added dropwise, and incubated for 1 hour at room temperature in the dark. Washing with PBS for 5 times; absorbing excessive PBS by using absorbent paper, adding an anti-fluorescence quencher (containing DAPI), incubating for 15min in a dark place, sealing by using a cover glass, observing the result under a laser confocal microscope, adjusting the laser intensity, keeping the contrast between groups consistent, and collecting images.
3. Results
As can be seen from comparison of pictures (fig. 1-2), the neural stem cell spheres of the experimental group are more compact and regular, spheres with large diameters can be obtained, meanwhile, the number of the spheres is more, the spherical state is more complete, and the neural spheres are more closely attached to the glass slide, so that the subsequent immunofluorescence test efficiency is higher.
In conclusion, the method of the invention can make the neural stem cell spheres more compact and regular, can obtain spheres with large diameter, and has the advantages of more spheres, more complete spherical state, better integrity of cell samples and more compact attachment on the glass slide, thereby improving the effect of immunofluorescence experiments.
Claims (9)
1. A cell sample preparation method for immunofluorescence experiments is characterized in that: the method comprises the following steps:
1) taking a cell culture, and replacing a culture medium in the cell culture with a PBS buffer solution;
2) cells were enriched onto slides using a centrifuge funnel;
3) air drying for 5-15 min.
2. The method of claim 1, wherein: the specific method for replacing in the step 1) is to remove the upper culture medium after centrifugation, add PBS buffer solution and resuspend.
3. The method of claim 2, wherein: the centrifugation parameters are as follows:
centrifuge at 100 Xg for 5 min.
4. The method of claim 1, wherein: the centrifugation parameters of the step 2) are as follows:
centrifuge at 400 Xg for 5 min.
5. The method of any of claims 1 to 4, wherein: the cell is a neural stem cell.
6. The method of claim 5, wherein: the neural stem cell is a neural stem cell sphere.
7. The method of claim 6, wherein: the neural stem cell ball is a rat neural stem cell ball.
8. The application of a cell sample in detecting protein in neural stem cells; the method is characterized in that:
the cell sample is prepared by the method according to any one of claims 1 to 7;
the application steps are as follows:
1) taking a cell sample, adding acetone or 4% paraformaldehyde for fixation, washing with a PBS buffer solution, adding a membrane penetrating solution and a sealing solution for incubation, removing the liquid, washing with the PBS buffer solution, and adding primary antibody for incubation;
2) and (3) taking the incubated liquid, washing with PBS buffer solution, adding a fluorescence-labeled secondary antibody for incubation, washing with PBS buffer solution, adding an anti-fluorescence quenching agent for incubation, and acquiring an image under a microscope.
9. The use of claim 8, wherein: the protein comprises Nestin and Sox2 protein.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020168657A1 (en) * | 2001-02-02 | 2002-11-14 | Chen Lan Bo | Rare event detection system |
| CN110376044A (en) * | 2019-08-29 | 2019-10-25 | 北京银丰鼎诚生物工程技术有限公司 | A kind of method of nerve ball immunofluorescence dyeing |
| CN210953565U (en) * | 2019-07-23 | 2020-07-07 | 第牛(上海)健康科技有限公司 | Cell centrifugal smear collector |
| CN111812071A (en) * | 2020-07-08 | 2020-10-23 | 中国医学科学院输血研究所 | Novel circulating tumor cell identification technology |
-
2021
- 2021-12-31 CN CN202111675599.5A patent/CN114317438A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020168657A1 (en) * | 2001-02-02 | 2002-11-14 | Chen Lan Bo | Rare event detection system |
| CN210953565U (en) * | 2019-07-23 | 2020-07-07 | 第牛(上海)健康科技有限公司 | Cell centrifugal smear collector |
| CN110376044A (en) * | 2019-08-29 | 2019-10-25 | 北京银丰鼎诚生物工程技术有限公司 | A kind of method of nerve ball immunofluorescence dyeing |
| CN111812071A (en) * | 2020-07-08 | 2020-10-23 | 中国医学科学院输血研究所 | Novel circulating tumor cell identification technology |
Non-Patent Citations (3)
| Title |
|---|
| 刘佳梅等: "一种获得神经干细胞单细胞的新方法", 中国康复理论与实, vol. 10, no. 1, pages 19 * |
| 周剑云等: "人脐带源神经干细胞的培养和分化", 中国康复理论与实践, vol. 18, no. 7, pages 615 - 618 * |
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