CN108254237A - A kind of live cell fluorescent colouring method - Google Patents
A kind of live cell fluorescent colouring method Download PDFInfo
- Publication number
- CN108254237A CN108254237A CN201711315361.5A CN201711315361A CN108254237A CN 108254237 A CN108254237 A CN 108254237A CN 201711315361 A CN201711315361 A CN 201711315361A CN 108254237 A CN108254237 A CN 108254237A
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- Prior art keywords
- cell
- living cells
- live cell
- fluorescent colouring
- cell fluorescent
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000004040 coloring Methods 0.000 title claims abstract description 15
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 claims abstract description 20
- 238000004043 dyeing Methods 0.000 claims abstract description 11
- 239000011521 glass Substances 0.000 claims abstract description 11
- 238000005406 washing Methods 0.000 claims abstract description 11
- 239000007853 buffer solution Substances 0.000 claims abstract description 10
- 239000006210 lotion Substances 0.000 claims abstract description 10
- 239000000644 isotonic solution Substances 0.000 claims abstract description 9
- 239000012128 staining reagent Substances 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 49
- 210000000130 stem cell Anatomy 0.000 claims description 19
- XDFNWJDGWJVGGN-UHFFFAOYSA-N 2-(2,7-dichloro-3,6-dihydroxy-9h-xanthen-9-yl)benzoic acid Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC(Cl)=C(O)C=C2OC2=CC(O)=C(Cl)C=C21 XDFNWJDGWJVGGN-UHFFFAOYSA-N 0.000 claims description 10
- VFNKZQNIXUFLBC-UHFFFAOYSA-N 2',7'-dichlorofluorescein Chemical group O1C(=O)C2=CC=CC=C2C21C1=CC(Cl)=C(O)C=C1OC1=C2C=C(Cl)C(O)=C1 VFNKZQNIXUFLBC-UHFFFAOYSA-N 0.000 claims description 5
- 239000004166 Lanolin Substances 0.000 claims description 4
- 229940039717 lanolin Drugs 0.000 claims description 4
- 235000019388 lanolin Nutrition 0.000 claims description 4
- 230000003647 oxidation Effects 0.000 claims description 4
- 238000007254 oxidation reaction Methods 0.000 claims description 4
- 239000000523 sample Substances 0.000 claims description 3
- 102000002322 Egg Proteins Human genes 0.000 claims 1
- 108010000912 Egg Proteins Proteins 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 210000004681 ovum Anatomy 0.000 claims 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 abstract description 14
- 235000011187 glycerol Nutrition 0.000 abstract description 7
- 230000001413 cellular effect Effects 0.000 abstract description 4
- 238000004925 denaturation Methods 0.000 abstract description 3
- 230000036425 denaturation Effects 0.000 abstract description 3
- 239000007850 fluorescent dye Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- 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
- G01N21/64—Fluorescence; Phosphorescence
Abstract
The present invention relates to biotechnology more particularly to a kind of live cell fluorescent colouring methods, include the following steps：（1）Living cells is dyed, 25 ~ 35min is cultivated in staining reagent；（2）After the completion of dyeing, living cells is transferred in washing lotion and is cleaned 2 ~ 5 times；（3）By the living cells cleaned on glass slide smear cell isotonic solution, coverslip in pressure；（4）It makes marks, uses confocal laser scanning microscope immediately.The present invention is improved innovation to conventional mounting method, replaces glycerine using cell isotonic solution DPBS buffer solutions in mounting, remains unchanged the cellular morphology on glass slide, overcomes the problem of cell shrinkage denaturation.
Description
Technical field
The present invention relates to biotechnology more particularly to a kind of live cell fluorescent colouring methods.
Background technology
Cell dyeing is an important technical for observing cell, during cell dyeing, generally requires much to grasp
Make step, using traditional cell dyeing method, be unfavorable for the Fluorescent Staining Observation of living cells.Cell after egg mother cell is fixed
Death has occurred, morphosis is fixed, then the step of progress fluorescent staining, and the step for being also finally most critical is envelope
Piece.Conventional egg mother cell mounting is that a thin layer of glycerine is applied on glass slide, and then egg mother cell is moved into glycerine, then
The coverslip that a small amount of lanolin is moistened with quadrangle carries out tabletting, and mounting is completed.But fluorescence dye is carried out for egg mother cell living
During color, mounting cannot mounting by a conventional method.After the completion of egg mother cell living is dyed with chemical dye, due to not solid
It is fixed, if continue with the method mounting cell of routine will crenation degeneration can even crush and seriously affect observation result.
Invention content
The problem of present invention is in order to overcome conventional cell dyeing to cause living cells crenation degeneration, influence observation, provides one
Kind live cell fluorescent colouring method.
To achieve these goals, the present invention uses following technical scheme：
A kind of live cell fluorescent colouring method, includes the following steps：
（1）Living cells is dyed, 25 ~ 35min is cultivated in staining reagent,
（2）After the completion of dyeing, living cells is transferred in washing lotion and is cleaned 2 ~ 5 times；
（3）By the living cells cleaned on glass slide smear cell isotonic solution, coverslip in pressure；
（4）It makes marks, uses confocal laser scanning microscope immediately.
The present invention is improved innovation to conventional mounting method, and glycerine is replaced using a kind of cell isotonic solution in mounting
The cellular morphology on glass slide is remained unchanged, overcomes the problem of cell shrinkage denaturation.
Preferably, the living cells is egg mother cell living.
Preferably, step（1）In, the method cultivated in staining reagent is：It is horizontal to the generation of ROS in egg mother cell
It is detected, with oxidation-sensitive fluorescence probe under 37 ~ 38 DEG C of temperature conditions, is incubated 25 ~ 35min.
Preferably, the oxidation-sensitive fluorescence probe is dichlorofluorescein DCFH, according to DCFH：The volume ratio of DPBS is
1：600 ~ 800 proportions are made.
Preferably, step（2）In, the washing lotion is DPBS washing lotions.
Preferably, step（3）In, the cell isotonic solution is DPBS buffer solutions.
Preferably, step（3）In, before covered, suitable lanolin is dipped in coverslip surrounding.
DPBS buffer solutions have salt balance, adjustable appropriate pH cushioning effect, are cell isotonic solution, in fluorescent staining
In for washing away the fluorescent dye of cell surface, so replacing glycerine, such energy with DPBS buffer solutions when living cells mounting
The morphosis and biological nature of cell are enough protected, and the lanolin that coverslip quadrangle is moistened with will be more than the lid of conventional mounting
Slide, progress ensure that cell is not crushed.
Therefore, the present invention has the advantages that：Innovation is improved to conventional mounting method, in mounting using thin
Born of the same parents isotonic solution DPBS buffer solutions replace glycerine, remain unchanged the cellular morphology on glass slide, overcome cell shrinkage denaturation
Problem.
Specific embodiment
Below by specific embodiment, the technical solutions of the present invention will be further described.
In the present invention, if not refering in particular to, all devices and raw material is commercially available or the industry is common are following
Method in embodiment is this field conventional method unless otherwise instructed.
Embodiment 1
（1）Egg mother cell living is dyed, the generation level of ROS in egg mother cell is detected, uses dichlorofluorescein
DCFH is incubated 25min, wherein DCFH under 37 DEG C of temperature conditions：The volume ratio of DPBS is 1：600；
（2）After the completion of dyeing, living cells is transferred in DPBS washing lotions and is cleaned 2 times；
（3）The living cells cleaned is smeared into DPBS buffer solutions on glass slide, coverslip in pressure；
（4）It makes marks, uses confocal laser scanning microscope immediately.
Embodiment 2
（1）Egg mother cell living is dyed, the generation level of ROS in egg mother cell is detected, uses dichlorofluorescein
DCFH is incubated 35min, wherein DCFH under 38 DEG C of temperature conditions：The volume ratio of DPBS is 1：800；
（2）After the completion of dyeing, living cells is transferred in DPBS washing lotions and is cleaned 5 times；
（3）The living cells cleaned is smeared into DPBS buffer solutions on glass slide, coverslip in pressure；
（4）It makes marks, uses confocal laser scanning microscope immediately.
Embodiment 3
（1）Egg mother cell living is dyed, the generation level of ROS in egg mother cell is detected, uses dichlorofluorescein
DCFH is incubated 30min, wherein DCFH under 37.5 DEG C of temperature conditions：The volume ratio of DPBS is 1：700；
（2）After the completion of dyeing, living cells is transferred in DPBS washing lotions and is cleaned 3 times；
（3）The living cells cleaned is smeared into DPBS buffer solutions on glass slide, coverslip in pressure；
（4）It makes marks, uses confocal laser scanning microscope immediately.
Comparative example
In comparative example, step（3）DPBS buffer solutions are substituted using glycerine, remaining step is identical with embodiment 1-3.
To being seen using the laser confocal microscope of comparative example and the live cell fluorescent colouring method of 1-3 of the embodiment of the present invention
It examines observation result to compare, the normal morphology of egg mother cell is spherical shape, is shot using the live cell fluorescent colouring method of comparative example
As a result it shows that egg mother cell has fold, illustrates that the form of cell membrane is denaturalized；Using the living cells of 1-3 of the embodiment of the present invention
Fluorescent staining method shooting result shows that egg mother cell is complete round, illustrates cellular morphology of the egg mother cell on glass slide
It remains unchanged, in normal state.
The foregoing is merely presently preferred embodiments of the present invention, not makees limitation in any form to the present invention, is not surpassing
There are other variants and remodeling under the premise of going out the technical solution recorded in claim.
Claims (7)
1. a kind of live cell fluorescent colouring method, which is characterized in that include the following steps：
（1）Living cells is dyed, 25 ~ 35min is cultivated in staining reagent,
（2）After the completion of dyeing, living cells is transferred in washing lotion and is cleaned 2 ~ 5 times；
（3）By the living cells cleaned on glass slide smear cell isotonic solution, coverslip in pressure；
（4）It makes marks, uses confocal laser scanning microscope immediately.
2. a kind of live cell fluorescent colouring method according to claim 1, which is characterized in that the living cells is ovum living
Mother cell.
A kind of 3. live cell fluorescent colouring method according to claim 2, which is characterized in that step（1）In, it is tried in dyeing
The method cultivated in agent is：The generation level of ROS in egg mother cell is detected, with oxidation-sensitive fluorescence probe in 37 ~ 38
Under DEG C temperature condition, it is incubated 25 ~ 35min.
4. a kind of live cell fluorescent colouring method according to claim 3, which is characterized in that the oxidation-sensitive fluorescence is visited
Needle is dichlorofluorescein DCFH, according to DCFH：The volume ratio of DPBS is 1：600 ~ 800 proportions are made.
5. according to a kind of any live cell fluorescent colouring methods of claim 1-4, which is characterized in that step（2）In, institute
Washing lotion is stated as DPBS washing lotions.
6. according to a kind of any live cell fluorescent colouring methods of claim 1-4, which is characterized in that step（3）In, institute
Cell isotonic solution is stated as DPBS buffer solutions.
7. according to a kind of any live cell fluorescent colouring methods of claim 1-4, which is characterized in that step（3）In, lid
Before upper coverslip, suitable lanolin is dipped in coverslip surrounding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711315361.5A CN108254237A (en) | 2017-12-12 | 2017-12-12 | A kind of live cell fluorescent colouring method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711315361.5A CN108254237A (en) | 2017-12-12 | 2017-12-12 | A kind of live cell fluorescent colouring method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108254237A true CN108254237A (en) | 2018-07-06 |
Family
ID=62721155
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711315361.5A Pending CN108254237A (en) | 2017-12-12 | 2017-12-12 | A kind of live cell fluorescent colouring method |
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| CN (1) | CN108254237A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109187146A (en) * | 2018-08-27 | 2019-01-11 | 青岛北大新世纪言鼎生物医学科技有限公司 | Human body cell holotype state immunofluorescence dyeing method and kit |
| CN110553888A (en) * | 2019-09-16 | 2019-12-10 | 中国农业科学院特产研究所 | Method for rapid tabletting of oocyte after immunofluorescence staining |
| CN112304731A (en) * | 2019-07-25 | 2021-02-02 | 中国农业科学院特产研究所 | Fluorescent staining method for porcine oocyte membrane protein NHE1 |
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| CN102879341A (en) * | 2012-09-29 | 2013-01-16 | 河北省农林科学院滨海农业研究所 | Method for identifying salt-resistant capability of cotton by using death rate of leaf lower epidermic cells |
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| CN101914490A (en) * | 2010-08-13 | 2010-12-15 | 中国医科大学 | Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof |
| CN102879341A (en) * | 2012-09-29 | 2013-01-16 | 河北省农林科学院滨海农业研究所 | Method for identifying salt-resistant capability of cotton by using death rate of leaf lower epidermic cells |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109187146A (en) * | 2018-08-27 | 2019-01-11 | 青岛北大新世纪言鼎生物医学科技有限公司 | Human body cell holotype state immunofluorescence dyeing method and kit |
| CN109187146B (en) * | 2018-08-27 | 2021-03-30 | 青岛言鼎生物医疗科技有限公司 | Human body cell full-form immunofluorescence staining method and kit |
| CN112304731A (en) * | 2019-07-25 | 2021-02-02 | 中国农业科学院特产研究所 | Fluorescent staining method for porcine oocyte membrane protein NHE1 |
| CN110553888A (en) * | 2019-09-16 | 2019-12-10 | 中国农业科学院特产研究所 | Method for rapid tabletting of oocyte after immunofluorescence staining |
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Application publication date: 20180706 |