CN112342264B - Double shift cell migration assay - Google Patents

Double shift cell migration assay Download PDF

Info

Publication number
CN112342264B
CN112342264B CN202011278717.4A CN202011278717A CN112342264B CN 112342264 B CN112342264 B CN 112342264B CN 202011278717 A CN202011278717 A CN 202011278717A CN 112342264 B CN112342264 B CN 112342264B
Authority
CN
China
Prior art keywords
slide
cell culture
cell
nipple
cell migration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202011278717.4A
Other languages
Chinese (zh)
Other versions
CN112342264A (en
Inventor
朱棠
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Putian University
Original Assignee
Putian University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Putian University filed Critical Putian University
Priority to CN202011278717.4A priority Critical patent/CN112342264B/en
Publication of CN112342264A publication Critical patent/CN112342264A/en
Application granted granted Critical
Publication of CN112342264B publication Critical patent/CN112342264B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry

Abstract

The invention discloses a twice-shift cell migration determination method, which belongs to the technical field of cell biology, and is characterized in that a nipple slide is placed in a culture hole of a first cell culture disc, cells to be detected are inoculated, the nipple slide is taken out after culture and placed in a culture hole of a second cell culture disc, and a cell culture solution and an experiment object are added; taking out the papillary slide, and quantifying by using an XTT colorimetric method or an MTT colorimetric method, wherein the papillary slide comprises a slide body and papillary protrusions arranged at the center of the slide body, and a plurality of through holes are formed in the slide body; the nipple slide is made of polymer resin. The twice shift cell migration measuring method is slightly influenced by manual operation, the cell migration distance is short, the cell migration distance is not easily influenced by cell proliferation, equipment used in a common laboratory is equipped, the required cost is low, and the common laboratory can design and complete the required cell migration measurement according to the twice shift cell migration measuring method provided by the invention.

Description

Double shift cell migration assay
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a twice-shift cell migration determination method.
Background
Cell migration assays are one of the basic methods for exploring the ability of cells to behave in cell biology studies. Are widely used in cell biology research. The mechanism principle of physiological and pathological processes, such as angiogenesis, embryonic development, immune cell function, tumor cell migration, wound healing and the like can be known through the research on the cell migration capability.
There are two methods currently used for cell migration assays. The first is wound healing experiment, which includes scraping one blank area of 1-2mm width of the cells on glass slide, marking the boundary with marker pen, culturing the glass slide in culture liquid for certain time, taking picture under microscope, analyzing the picture, calculating the area occupied by the migrated cells and the total area of the blank area to obtain cell migration rate. Because the boundary needs to be manually drawn, and the microscopic analysis is adopted, the problems of low efficiency, large influence of human factors, easy fatigue in the analysis process, low operation repeatability, large deviation, capability of measuring only partial cell migration and incapability of measuring all cell migration exist. The second is a filtration membrane migration test (Boyden chamber method) in which cells are cultured in a porous polycarbonate membrane cup, after a certain time, the migrated cells pass through the membrane to the opposite side of the membrane cup, the non-migrated cells remain in the cup, the non-migrated cells in the cup are scraped off with a cotton swab, and then staining, fluorescent staining, or 35 S]And labeling the migrated cells left on the reverse side of the membrane by using a method such as methionine isotope labeling, and then quantitatively analyzing the amount of the migrated cells passing through the filtration membrane. Because the cells remained in the cup need to be manually erased, the time is long, and the problem of incomplete erasing cannot be avoided,meanwhile, the price of the porous polycarbonate membrane cup is high, the number of the required porous polycarbonate membranes is large during experiments, and the cost is high. The two methods usually measure the influence caused by cell proliferation because the migration process needs to be cultured for 2-3 days.
Disclosure of Invention
In order to overcome the defects of the prior art, the technical problems to be solved by the invention are as follows: how to provide a cell migration assay method which is low in cost, accurate in measurement and not easily affected by manual operation.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for two shift cell migration assay comprising the steps of:
step 1, placing a nipple slide in a culture hole of a first cell culture disc, and then inoculating a cell to be detected for cell culture;
step 2, taking out the nipple slide after the cell culture is finished, placing the nipple slide in a culture hole of a second cell culture disc, adding an observation experiment object, and culturing for 12-24 hours;
step 3, taking out the nipple slide, and quantifying the cells in the culture wells of the second cell culture disc by using an XTT colorimetric method or an MTT colorimetric method;
the nipple glass slide comprises a glass slide body and a mastoid arranged at the center of the glass slide body, and a plurality of through holes are formed in the glass slide body; the nipple glass sheet is made of polymer resin.
The invention has the beneficial effects that: the twice-displacement cell migration determination method provided by the invention realizes clear delimitation, effectively separates in-situ cells from migration cells through twice displacement, shortens the cell migration distance by utilizing through holes distributed at multiple positions of a nipple slide, can finish migration within 24h, effectively avoids errors caused by cell proliferation, simultaneously automatically analyzes and evaluates the cell migration rate by introducing XTT (or MTT) staining through a spectrophotometer, simplifies the experimental steps, eliminates manual delimitation, manually removes in-situ cells, and manually delimits the cell movement area through a microscope for calculation and evaluation, thereby solving the problems of time consumption, fatigue and low repetition rate.
Drawings
FIG. 1 is a schematic view showing a partial structure of a papillary slide and a cell culture dish according to an embodiment of the present invention;
FIG. 2 is an enlarged view of FIG. 1 at A;
FIG. 3 is a partial schematic view of a papillary slide and cell culture dish according to an embodiment of the present invention;
FIG. 4 is an enlarged view of FIG. 3 at B;
description of reference numerals: 1. a papillary slide; 11. a through hole; 12. mastoid; 13. a groove; 2. culture wells of cell culture plates.
Detailed Description
In order to explain the technical contents, the objects and the effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
Referring to fig. 1 to 4, the present invention provides a method for measuring migration of two displaced cells, comprising the following steps:
step 1, placing a nipple slide in a culture hole of a first cell culture disc, and then inoculating a cell to be detected for cell culture;
step 2, taking out the papillary slide after cell culture is finished, placing the papillary slide in a culture hole of a second cell culture disc (the first cell culture disc and the second cell culture disc are cell culture discs with the same specification, namely porous cell culture discs used in common laboratories), finishing first displacement, then adding cell culture solution and an experimental substance (such as a chemical substance, a polypeptide, a bioactive substance inhibitor or an excitant), and culturing for 12-24h, wherein cells migrate;
step 3, taking the nipple slide out of the second cell culture disc, completing the second displacement, taking the cells left in the second cell culture disc as migration cells, staining the cells by using XTT or MTT, then placing the second cell culture disc in an automatic spectrophotometer, and quantifying the cells in the second cell culture disc by using an XTT colorimetric method or an MTT colorimetric method;
the nipple glass slide comprises a glass slide body and a mastoid arranged at the center of the glass slide body, and a plurality of through holes are formed in the glass slide body; the nipple glass sheet is made of polymer resin.
From the above description, the beneficial effects of the present invention are: the twice-displacement cell migration determination method provided by the invention realizes clear delimitation, effectively separates in-situ cells from migration cells through twice displacement, shortens the cell migration distance by utilizing through holes distributed at multiple positions of a nipple slide, can finish migration within 24h, effectively avoids errors caused by cell proliferation, simultaneously automatically analyzes and evaluates the cell migration rate by using an XTT (or MTT) staining through a spectrophotometer, simplifies the experimental steps, eliminates manual delimitation, manually eliminates in-situ cells, and solves the problems of time consumption, fatigue, low repetition rate and large human errors caused by calculating and evaluating the manually-defined cell movement area by a microscope. The twice shift cell migration determination method provided by the invention is not easily influenced by manual operation, the culture time in the cell migration process is short, the twice shift cell migration determination method is not easily influenced by cell proliferation, meanwhile, the used equipment automatic spectrophotometer is equipped in a common laboratory, the required cost is low, and the common laboratory can automatically design and complete the required cell migration determination according to the twice shift cell migration determination method provided by the invention and a nipple slide.
Furthermore, the nipple slide is cylindrical or cuboid. The shape of the papillary slide is not particularly limited, and the papillary slide may be placed in the culture well of the cell culture dish.
Further, the shape of the through hole is a strip, a circle, a rectangle, a parallelogram or a triangle. The shape of the through-hole can be arbitrarily selected and can be other shapes than the above-listed types.
Further, the thickness of the nipple slide is 0.1-0.5 mm. The thickness of the papillary slide should not be too thick, which may affect the migration of cells.
Furthermore, the mastoid is composed of a hemispherical upper part and a cylindrical lower part, a groove is arranged at the center of the upper part, and the upper part and the lower part are integrally formed. The nipple slide is convenient to be clamped by tweezers and does not touch cells on the nipple slide.
Furthermore, the mastoid is a columnar structure with a narrow middle part and wide two sides. The forceps can clamp the middle part of the mastoid, and because the two sides are wide, the forceps can not slide down, so that the forceps can conveniently clamp the papillary slide and can not touch or pollute cells on the papillary slide.
Further, the papilla of the nipple slide is clamped and moved by using the disinfected forceps in the step 2. The tweezers need to be used for clamping the papillary slide after being burnt and disinfected on the alcohol lamp, so that pollution is avoided.
Furthermore, the models of the first cell culture disc and the second cell culture disc are the same, and the first cell culture disc is a 24-hole cell culture disc, a 48-hole cell culture disc or a 96-hole cell culture disc.
Example 1:
referring to FIGS. 1-2, a two-shift cell migration assay method specifically includes the steps of:
step 1, placing the papilla slide in culture holes of a first 96-hole cell culture disc in sequence, then inoculating 100 mu L of cells to be detected into each hole (culture hole of the culture disc) for cell culture, and forming 2-4x10 the next day 4 Density of individual cells/well;
step 2, taking out the papillary slides one by one after the cell culture is finished (the cell culture is full of 80-100%), clamping the papillary slides one by using sterilized forceps, putting the papillary slides into culture holes of a second 96-hole cell culture disc one by one, finishing the first displacement, then adding cell culture solution and an experimental substance (such as organic reagent, inorganic reagent, polypeptide, bioactive substance inhibitor, stimulant and the like), culturing for 12-24h, wherein cells migrate, and the migrated cells leave the papillary slides to the bottom disc of the culture holes of the second cell culture disc through the edges and through holes of the papillary slides;
step 3, taking out and discarding the nipple slide from the second 96-well Cell culture disc, completing the second displacement, adding 50 mu XTT determination working solution (TACS XTT Cell promotion/visualization Assay, available in Shanghai river Qu professional operation laboratory reagent) into each well of the second 96-well Cell culture disc to stain the cells, then placing the second 96-well Cell culture disc into an automatic spectrophotometer, and quantifying the cells in the second 96-well Cell culture disc by using an XTT colorimetric method;
the nipple glass slide comprises a glass slide body and a mastoid arranged at the center of the glass slide body, and a plurality of through holes are formed in the glass slide body; the through hole is circular; the nipple glass sheet is made of polyvinyl chloride resin;
the papillary slide is cylindrical, the diameter of the papillary slide is smaller than that of a culture hole of a 96-hole cell culture disc, and the thickness of the papillary slide is 0.1 mm;
the mastoid is composed of a hemispherical upper part and a cylindrical lower part, a groove is formed in the center of the hemispherical upper part of the mastoid, and the upper part and the lower part are integrally formed.
Example 2:
referring to fig. 3-4, a two-shift cell migration assay method specifically includes the steps of:
step 1, placing a papilla slide in a culture hole of a first 48-hole cell culture disc, then inoculating 200 mu L of cells to be detected into each hole (culture hole of the culture disc), and carrying out cell culture to form 6-8x10 on the next day 4 Density of individual cells/well;
step 2, taking out the papillary slides one by one after cell culture is finished, sequentially placing 48 papillary slides in culture holes of a second 48-hole cell culture disc to finish first displacement, then adding cell culture solution and an experimental substance (such as an organic reagent, an inorganic reagent, polypeptide, a bioactive substance inhibitor, an excitant and the like), and culturing for 12-24h, wherein cells migrate;
step 3, taking the papilla slide out of the second 48-hole Cell culture disc, completing the second displacement, namely, obtaining the migration cells remained in the second Cell culture disc, adding 50 mu of MTT determination working solution (TACS MTT Cell promotion/visualization Assay, Shanghai river Qu reaches the reagent of professional management laboratories) into each hole of the second 48-hole Cell culture disc, dyeing the cells, then taking 40 mu of dyeing solution into an automatic spectrophotometer, and quantifying the cells in the second 48-hole Cell culture disc by utilizing an MTT colorimetric method;
the nipple glass slide comprises a glass slide body and a mastoid arranged at the center of the glass slide body, and a plurality of through holes are formed in the glass slide body; the through hole is rectangular; the nipple glass sheet is made of epoxy resin;
the papillary slide is cylindrical, the diameter of the papillary slide is smaller than that of a culture hole of a 48-hole cell culture disc, and the thickness of the papillary slide is 0.3 mm;
the mastoid is a columnar structure with a narrow middle and wide two sides.
Example 3:
example 3 differs from example 1 in that:
the first cell culture disc and the second cell culture disc are both 24-hole cell culture discs;
the nipple glass sheet is made of polyethylene resin;
the through-hole is the bar, the thickness of nipple slide is 0.5mm, nipple slide is the cuboid form, nipple slide's length is less than the diameter in the cultivation hole of 24 hole cell culture dishes.
Example 4:
example 4 differs from example 2 in that:
the nipple glass sheet is made of polypropylene resin;
the through-hole is the rhombus, the thickness of nipple slide is 0.25mm, nipple slide is the cuboid form, nipple slide's diameter is less than the diameter of 48 hole cell culture dish's cultivation hole.
In conclusion, the twice-shift cell migration determination method provided by the invention realizes clear delimitation, effectively separates in-situ cells from migration cells through twice shifts, shortens the cell migration distance by using a papillary slide and through holes distributed at multiple positions, can finish migration within 24h, effectively avoids errors caused by cell proliferation, simultaneously automatically analyzes and evaluates the cell migration rate by a spectrophotometer by introducing XTT (or MTT) staining, simplifies the experimental steps, eliminates the problems of time consumption, fatigue and low repetition rate caused by manual delimitation, manual removal of in-situ cells and manual planning of the cell movement area by a microscope for calculation and evaluation.
The above description is only an embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention and the contents of the accompanying drawings, which are directly or indirectly applied to the related technical fields, are included in the scope of the present invention.

Claims (8)

1. A method for measuring migration of displaced cells twice, comprising the steps of:
step 1, placing a nipple slide in a culture hole of a first cell culture disc, and then inoculating a cell to be detected for cell culture;
step 2, taking out the nipple slide after the cell culture is finished, placing the nipple slide in a culture hole of a second cell culture disc, adding cell culture solution and an experimental object, and culturing for 12-24 hours;
the experimental substance is an organic reagent and/or an inorganic reagent;
step 3, taking out the nipple slide, and quantifying the cells in the second cell culture disc by using an XTT colorimetric method or an MTT colorimetric method;
the papillary slide comprises a slide body and a papilla arranged at the center of the slide body, wherein a plurality of through holes are formed in the slide body; the nipple glass sheet is made of polymer resin.
2. The method for measuring migration of cells by double shift according to claim 1, wherein the papillary slide has a cylindrical shape or a rectangular parallelepiped shape.
3. The method of two shift cell migration assays of claim 1, wherein the shape of the through-hole is a bar, a circle, a rectangle, a parallelogram, or a triangle.
4. The method for two shift cell migration assays according to claim 1, wherein the thickness of the papillary slide is 0.1-0.5 mm.
5. The method for measuring migration of two displaced cells according to claim 1, wherein the mastoid is composed of a hemispherical upper portion and a cylindrical lower portion, the upper portion has a recess at the center thereof, and the upper portion and the lower portion are integrally formed.
6. The method for two shift cell migration assays according to claim 1, wherein the mastoid is a columnar structure with a narrow middle and wide sides.
7. The method for two-shift cell migration assay according to claim 1, wherein the step 2 is performed by moving the papilla slide by using sterilized forceps to clamp the papilla of the papilla slide.
8. The method of two-pass shift cell migration assay of claim 1, wherein said first cell culture tray is a 24-well cell culture tray, a 48-well cell culture tray, or a 96-well cell culture tray.
CN202011278717.4A 2020-11-16 2020-11-16 Double shift cell migration assay Active CN112342264B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202011278717.4A CN112342264B (en) 2020-11-16 2020-11-16 Double shift cell migration assay

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011278717.4A CN112342264B (en) 2020-11-16 2020-11-16 Double shift cell migration assay

Publications (2)

Publication Number Publication Date
CN112342264A CN112342264A (en) 2021-02-09
CN112342264B true CN112342264B (en) 2022-08-26

Family

ID=74364001

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011278717.4A Active CN112342264B (en) 2020-11-16 2020-11-16 Double shift cell migration assay

Country Status (1)

Country Link
CN (1) CN112342264B (en)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003216058A1 (en) * 2002-01-14 2003-07-30 The Board Of Trustees Of The University Of Illinois Mammalian neural stem cells, compositions and uses thereof
CN104830681A (en) * 2014-02-09 2015-08-12 李保伟 Cell culture glass slide system
CN104774763A (en) * 2015-04-24 2015-07-15 何向锋 Microchip type single cell cloning separation culture plate and single cell cloning separation method
IT201800001123A1 (en) * 2018-01-16 2019-07-16 Rigenerand S R L A DEVICE AND A METHOD FOR THREE-DIMENSIONAL CELLULAR CULTURE
CN110684818A (en) * 2019-09-05 2020-01-14 福建省医学科学研究院 Application of real-time unmarked cell analyzer in detecting proliferation and migration capacity of esophageal cancer cells

Also Published As

Publication number Publication date
CN112342264A (en) 2021-02-09

Similar Documents

Publication Publication Date Title
CN103257213B (en) A kind of fully integrated high-flux cell horizontal micro-fluidic chip drug evaluation system
US10940476B2 (en) Device for high-throughput multi-parameter functional profiling of the same cells in multicellular settings and in isolation
CN201737950U (en) Special experimental apparatus for cell growing on cover glass
CN112424329A (en) Systems and methods for cell imaging, analysis, and measurement
CN112342264B (en) Double shift cell migration assay
US3989591A (en) Test method for separating and/or isolating bacteria and tissue cells
US20130281323A1 (en) Device and method for cell-exclusion patterning
CN116593262B (en) Molecular marker detection product based on PDX/PDTX tumor living tissue biological sample and database and preparation method thereof
CN110461244A (en) For handling, detecting and the Multiple-Aperture Device of the biomaterial of paraffin or plastic embedding (IFPE) that multiple analysis is completely fixed
CN202562928U (en) Visualized protein chip device
CN108351282B (en) Methods for cell counting and viability measurement
US20240018452A1 (en) Chip for integrated tumor cell behavior experiments
US4200056A (en) Segmented platen for applying liquids to a flat surface
CN202201884U (en) Kit for cell biotechnology
CN114088501A (en) Chip device for in-situ tissue staining and decoloring and use method
CN104073433A (en) Large quantitatively counting microscope detection glass slide for microorganisms
CN217466393U (en) Sample preparation device for morphological detection
RU105744U1 (en) DEVICE FOR MICROBIOLOGICAL RESEARCH
CN219641355U (en) Flow cytometer sample preprocessor
RU2809851C1 (en) Cassette for system for crystallization of membrane proteins for purposes of on-line x-ray structural analysis
CN219807967U (en) Auxiliary structure for monoclonal antibody subcloning of erythrocyte membrane
CN216550482U (en) Cell glass slide climbing device for observation and detection
CN220084477U (en) Microorganism staining rack
CN2480842Y (en) Uropsammus quantitative analysis instrument
CN216210215U (en) Novel glass slide

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant