CN108384715B - Culture dish for cell scratch experiment and use method thereof - Google Patents
Culture dish for cell scratch experiment and use method thereof Download PDFInfo
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- CN108384715B CN108384715B CN201810390218.0A CN201810390218A CN108384715B CN 108384715 B CN108384715 B CN 108384715B CN 201810390218 A CN201810390218 A CN 201810390218A CN 108384715 B CN108384715 B CN 108384715B
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Abstract
The invention relates to a culture dish for cell scratch experiments and a use method thereof, wherein the culture dish comprises a culture dish body and a round cover clamping layer, wherein the round cover clamping layer comprises an inverted hook ring, a cell scratch plate and a plurality of connecting rods for connecting the inverted hook ring and the cell scratch plate; the barb is hung on the edge of the culture dish body in a ring manner; the cell scratch board is arranged in the culture dish body and is formed by intersecting at least one transverse glass partition board and at least one longitudinal glass partition board, the lower parts of two side surfaces of the glass partition boards incline inwards, and the bottom surface of the glass partition board is tightly attached to the bottom surface of the culture dish body. When the culture dish is used for cell scratch experiments, the interlayer can be directly placed or removed in a handheld mode, pollution is avoided, regular cell scratches with uniform width and high straightness are conveniently obtained, the experimental efficiency can be effectively improved, the experimental cost is reduced, and the accuracy of experimental data is improved.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a culture dish for cell scratch experiments and a use method thereof.
Background
The cell scratch test is an in vitro test method for researching cell migration/tumor invasiveness. The principle is that when cells grow to be fused into a single-layer state, a blank area called a scratch is artificially manufactured on the fused single-layer cells, and the cells at the edge of the scratch gradually enter the blank area to heal the scratch.
The basic steps of cell scratch experiments include the manufacture of "scratches", the acquisition of images during cell migration, and the processing of later data. The manufacturing process of scratches is a key to influence experimental results, and the main method at present is to make cells in a region where a gun tip passes fall off by linearly passing through a gun head of a pipette perpendicular to the surface of a cell culture dish, so that a cell-free region is manufactured, namely a scratch. The width of the score depends on the width of the gun tip. After the scratch is made, cells on both sides of the blank area migrate to the middle and gradually cover the "scratch". The ability of tumor cells to migrate to the empty region reflects their ability to invade, metastasize, and can be quantitatively measured using the area of the empty region. The more aggressive the cell, the faster the scratch area shrinks. Therefore, the change of the cell invasion capacity of different treatment groups can be compared by accurately measuring the trend of the scratch area along with the time, and the method has important significance in the fields of cancer occurrence mechanism, tumor treatment and the like.
Because of the working and material reasons of the pipette gun heads, the gun tip width variation is larger before and after different gun heads or the same gun head are used; in addition, the cell scratch width is in the mu m level, and the small difference can cause the doubled area change, so that the force and angle of an operator using a gun head to scratch the surface of the culture dish can seriously influence the scratch area; secondly, the edges of scratches formed by external force scratch are in a zigzag shape, and a standard straight line is difficult to achieve, so that inconvenience is caused by area calculation, and errors are easy to generate; again, when counting scratch areas, it is often necessary to observe several time points under a microscope, for example, scratch generation is performed for 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, etc., and the scratch areas of the experimental group and the control group at each time point are counted separately, and strictly speaking, each time data acquisition should come from the same area, but in actual operation, it is difficult for an operator to accurately identify the area of the last data acquisition in a highly similar cell group due to inconvenient operation under the microscope and rapid migration of cells; in addition, the whole scratching operation is operated above the culture medium and is directly contacted with the culture medium, and bacterial pollution is easily caused when the culture medium is placed and taken out. These factors can cause significant experimental errors, lead to erroneous conclusions, or lead to reduced reproducibility of the experiment. Accordingly, there is a need in the art for a cell score plate that ensures high straightness of the score, good width uniformity, and ease of handling.
Disclosure of Invention
Aiming at the technical problems of low straightness and width uniformity of cell scratches in the prior art, the invention aims to provide a culture dish for cell scratch experiments, which can ensure high straightness of scratches, good width uniformity and convenient operation.
The culture dish for the cell scratch experiment comprises a culture dish body and is characterized by further comprising a round cover clamping layer, wherein the round cover clamping layer comprises an inverted hook ring, a cell scratch plate and a plurality of connecting rods for connecting the inverted hook ring and the cell scratch plate;
the barb ring is hung on the edge of the culture dish body, a clamping groove is formed in the outer side and/or the inner side of the edge of the culture dish body, and a buckle corresponding to the clamping groove is arranged on the inner side wall of the barb ring;
the cell scratch board is arranged in the culture dish body and consists of at least one transverse glass partition board and at least one longitudinal glass partition board in a crossing way, the lower parts of two side surfaces of the glass partition boards incline inwards, namely, the glass partition boards gradually narrow from the middle part to the bottom surface to be conical, the bottom surface of the glass partition boards is tightly attached to the bottom surface of the culture dish body, and the bottom surface width of the glass partition boards is 400-600 mu m, preferably 500 mu m.
According to the invention, when the cell scratch board is arranged in the culture dish body, the strip-shaped bottom surface of the glass partition board is tightly attached to the bottom surface of the culture dish body, and the barb ring is hung on the edge of the culture dish body, so that pressure can be transmitted to the cell scratch board through the connecting rod on the one hand, the cell scratch board is further clung to the bottom surface of the culture dish body, on the other hand, the limiting effect is also achieved, and the cell scratch board can be prevented from sliding. When the cell scratch experiment is carried out, cell suspension is added, after the cell fusion degree reaches 80% or more, the round cover clamping layer is vertically and upwards removed, at the moment, a cell blank area with uniform width and high straightness corresponding to the glass partition plate, namely a scratch, can appear at the bottom of the culture dish body, cells are continuously cultured after the serum-free culture medium is replaced, and the healing condition of the scratch is observed. One of the advantages of the culture dish sandwich design is that the inverted hook ring does not need to keep strict sterility, can be directly taken and placed by hand, and is convenient and quick to operate.
In the invention, the lower parts of the two side surfaces of the glass partition board incline inwards, namely the width of the glass partition board gradually narrows from the middle part to the bottom surface to form a cone shape, and the design can effectively prevent cells from growing on the two side surfaces of the glass partition board, and damage scratches caused by the traction can be avoided when the cell scratch board is removed.
The clamping groove at the edge of the culture dish body can be designed into a whole annular closed groove along the edge of the culture dish body, and a plurality of grooves with four preferable intervals can be symmetrically arranged; the buckles on the inner side wall of the barb ring are correspondingly arranged.
The glass separators can be crossed vertically or obliquely, and can be crossed to form a shape of '十', '工', '王', '卄', '卅', '井', and the like. Scratches formed through the glass separator will create intersections that can provide a location for taking pictures under a microscope.
In a preferred embodiment of the present invention, the cell scratch board is formed by vertically crossing two transverse glass partition boards and two longitudinal glass partition boards, and is in a shape of a Chinese character 'jing'. The cell scratch board is in a 'well' shape, and particularly scratches formed by the cell scratch board have four crossing points, so that the cell scratch board is more convenient to position when observed by a microscope.
Further, the connecting rods are four in number, one ends of the four connecting rods are connected with the barb rings, and the other ends of the four connecting rods are respectively connected with the outer sides of the four crossing points of the glass partition plate.
Preferably, the glass separators are equidistantly distributed.
Preferably, the length of the glass separator is 0.8-3.8cm.
Preferably, the connecting rod is spliced, clamped or integrally connected with the cell scratch board. When the movable connection modes such as plugging or clamping are used, the inverted hook ring can be reused, and the cell scratch board can be replaced by selecting different shapes according to the requirement of no scratch.
In a preferred embodiment of the present invention, the cross section of the glass partition plate is diamond, the upper part and the lower part of the glass partition plate are symmetrical, the glass partition plate is gradually narrowed from the middle part to the top surface to be tapered, and the top surface width is 400-600 μm, preferably 500 μm. This design allows the cell score plate to be used on both sides.
A preferred embodiment of the invention comprises a dish cover, wherein the shape of the inner side wall of the dish cover corresponds to the shape of the barb ring.
The invention also provides a use method of the culture dish for cell scratch experiments, which comprises the following steps:
a) Performing high-pressure steam sterilization on the culture dish for cell scratch experiments in advance, and clamping the barb ring with the circular cover interlayer on the edge of the culture dish body to enable the cell scratch plate to be tightly attached to the bottom surface of the culture dish body;
b) Adding a proper amount of cell suspension into the culture dish body, and placing the culture dish body into an incubator for culture;
c) And after the culture is carried out for a preset period of time, observing the cell adherence condition and the cell density, removing the circular cover clamping layer when the cell fusion degree reaches more than 80%, replacing the serum-free culture medium, continuously culturing, and observing the scratch healing condition.
The invention has the advantages that:
1. in the invention, the strip-shaped bottom surface of the glass partition plate can be tightly attached to the bottom surface of the culture dish, and cells cannot reach the attaching part. Simultaneously, the lower part of the both sides face of glass baffle inwards inclines, promptly the glass baffle is narrowed gradually and is the toper from middle part to bottom surface width, and this design can effectively prevent that the cell from sticking to the both sides face growth of glass baffle can avoid appearing the involvement and damage the mar when removing the cell mar board.
2. The barb ring is hung on the edge of the culture dish body, on one hand, the pressure can be transmitted to the cell scratch board through the connecting rod, the cell scratch board is further enabled to cling to the bottom surface of the culture dish body, on the other hand, the barb ring also plays a limiting role, and the cell scratch board can be prevented from sliding. The design can effectively fix the cell scratch board, avoid the cell scratch board sliding when transferring the culture dish, ensure the regularity and straightness of scratches, and simultaneously not affect the growth parts of other cells of the culture dish. Moreover, the barb ring does not need to keep strict sterility, can be directly taken and placed by hand, and is convenient and quick to operate.
3. When the culture dish for cell scratch experiments is used for experiments, each scratch can be directly used for the experiments, so that the scratch obtained by repeatedly using the gun head is avoided, the experimental efficiency is improved, the experimental cost is reduced, and the accuracy of the obtained experimental data is high.
4. The preferred embodiment of the invention adopts the cell scratch board in the shape of the Chinese character 'jing', can provide positioning for photographing cells under a microscope, and is more convenient.
Drawings
FIG. 1 is a schematic diagram showing the composition of a culture dish for cell streaking experiments according to the present invention;
FIG. 2 is a schematic bottom view of a circular cap sandwich of the present invention;
FIG. 3 is a schematic illustration of the snap fit of the circular cap sandwich and culture dish body of the present invention;
FIG. 4 is a schematic view of a cell scratch pad of the present invention.
Reference numerals
A culture dish body 1 and a clamping groove 11; the round cover clamping layer 2, the inverted hook ring 21, the cell scratch plate 22, the side surface 221, the bottom surface 222, the connecting rod 23 and the buckle 24; and a dish cover 3.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1 Petri dish for cell scratch experiments
FIG. 1 shows a culture dish for cell streaking experiments according to a preferred embodiment of the present invention, which comprises a culture dish body 1, a circular cap-mounting interlayer 2 and a dish cap 3.
As shown in fig. 2, the circular cap holder 2 includes a reverse hook ring 21, a cell scratch plate 22, and four connection bars 23. Wherein, as shown in FIG. 3, the reverse hook ring 21 can be hung on the edge of the culture dish body 1, and the cell scratch plate 22 can be attached to the bottom surface of the culture dish body 1. Four clamping grooves 11 are symmetrically formed in the outer side of the edge of the culture dish body 1, and buckles 24 corresponding to the clamping grooves 11 are arranged on the inner side wall of the barb ring 1. As shown in FIG. 4, the cell scratch board 22 is formed by vertically crossing two transverse and two longitudinal glass partition boards into a 'well' -shape, the cross section of the glass partition board is diamond-shaped, the width of each of the two side faces 221 from the middle part to the top face and the bottom face 222 is gradually narrowed to be conical, and the width of the top face and the bottom face 222 is 500 mu m; the width of the middle part of the glass partition plate is maximum and is 2mm. The glass separator was 9mm long. One end of each of the four connecting rods 23 is connected with the inverted hook ring 21, and the other end is connected with the outer sides of the four crossing points of the glass partition plate.
When cell scratch board 22 is arranged in culture dish body 1, the rectangular bottom surface 222 of glass baffle closely laminates with culture dish body 1 bottom surface, and barb ring 21 hangs on culture dish body 1 edge, can transmit pressure to cell scratch board 22 through connecting rod 23 on the one hand, further makes cell scratch board 22 hug closely with the bottom surface of culture dish body 1, on the other hand still plays spacing effect, can avoid cell scratch board 22 to slide.
In this embodiment, when cell scratch board 22 is arranged in culture dish body 1, the rectangular bottom surface 222 of glass baffle closely laminates with culture dish body 1 bottom surface, and barb ring 21 hangs on culture dish body 1 edge accessible connecting rod 23 on the one hand can be to cell scratch board 22 transmission pressure, further makes cell scratch board 22 hug closely with the bottom surface of culture dish body 1, and on the other hand still plays spacing effect, can avoid cell scratch board 22 to slide. In addition, the reverse hook ring 21 does not need to keep strict sterility, can be directly taken and put by hand, and is convenient and quick to operate.
Specifically, when the culture dish for cell streak test of example 1 is used for cell streak test, the following procedure is followed:
a) Sterilizing the culture dish for cell scratch experiments by high-pressure steam in advance, clamping the reverse hook ring 21 of the round cover interlayer 2 on the edge of the culture dish body 1, enabling the cell scratch plate 22 to be clung to the bottom surface of the culture dish body 1, and then placing the culture dish in a sterile area for later use;
b) Treating and collecting adherent cells by trypsin, washing by phosphate buffer solution and centrifuging, and re-suspending the cells with a preset amount of culture medium; determining the cell density in suspension; taking a proper amount of cells in another centrifuge tube, diluting with a preset amount of culture medium, adjusting the cell density to 6000-20000/mL, taking 400-500 mu L of cell suspension, adding into the culture dish body 1, and then placing into a cell culture box to avoid shaking and tilting;
c) After culturing for a preset period of time, observing the cell adherence condition and the cell density, and when the cell fusion degree reaches more than 80%, vertically removing the circular cover clamping layer 2 upwards, wherein at the moment, a cell blank area with uniform width and high straightness corresponding to the glass partition plate, namely a scratch, appears at the bottom of the culture dish body 1; then the culture medium without serum is replaced for continuous culture, and the healing condition of scratches is observed.
In this embodiment, the lower parts of the two sides 221 of the glass partition board are inclined inwards, that is, the glass partition board gradually narrows from the middle part to the bottom to be conical, so that the design can effectively prevent cells from growing on the two sides of the glass partition board, and damage to scratches caused by the connection of the cells can be avoided when the cell scratch board is removed.
In this embodiment, the scratch formed by cell scratch pad 22 is also "well" shaped, having four points of intersection that provide positioning for microscopic photography, making positioning more convenient for microscopic viewing.
In the above embodiment, the reverse hook ring 21, the cell scratch plate 22 and the connection rod 23 are integrally constructed. Of course, they can also be movably connected by plugging or clamping, for example, the cell scratch board 22 and the connecting rod 23 can be movably connected, the inverted hook ring 21 can be reused, and the cell scratch board 22 can be replaced by selecting different shapes according to the need of no scratch.
While the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.
Claims (3)
1. The culture dish for the cell scratch experiment comprises a culture dish body and is characterized by further comprising a round cover clamping layer, wherein the round cover clamping layer comprises an inverted hook ring, a cell scratch plate and a plurality of connecting rods for connecting the inverted hook ring and the cell scratch plate;
the barb ring is hung on the edge of the culture dish body, a clamping groove is formed in the outer side and/or the inner side of the edge of the culture dish body, and a buckle corresponding to the clamping groove is arranged on the inner side wall of the barb ring;
the cell scratch board is arranged in the culture dish body and consists of at least one transverse glass partition board and at least one longitudinal glass partition board which are crossed, wherein the lower parts of the two side surfaces of the glass partition board incline inwards, namely the glass partition board gradually narrows from the middle part to the bottom surface to be conical, the bottom surface of the glass partition board is tightly attached to the bottom surface of the culture dish body, and the bottom surface width of the glass partition board is 400-600 mu m;
the cell scratch board is formed by vertically crossing two transverse glass partition boards and two longitudinal glass partition boards, and is in a shape of a Chinese character 'jing';
the four connecting rods are arranged in total, one ends of the four connecting rods are connected with the barb rings, and the other ends of the four connecting rods are respectively connected with the outer sides of the four crossing points of the glass partition plate;
the glass partition plate is provided with an arched handle, and two ends of the arched handle are connected to two intersection points of opposite angles of the glass partition plate;
the length of the glass separator is 0.8-3.8cm;
the connecting rod is spliced, clamped or integrally connected with the cell scratch board;
the cross section of the glass partition plate is diamond-shaped, the upper part and the lower part of the glass partition plate are symmetrical, the width of the glass partition plate from the middle part to the top surface is gradually narrowed to be conical, and the width of the top surface is 400-600 mu m.
2. The culture dish for cell streaking experiments as in claim 1 further including a dish cover having an inner side wall shape corresponding to the barb ring shape.
3. The method of using a culture dish for cell scratch test according to any one of claims 1 to 2, comprising the steps of:
a) Performing high-pressure steam sterilization on the culture dish for cell scratch experiments in advance, and clamping the barb ring with the circular cover interlayer on the edge of the culture dish body to enable the cell scratch plate to be tightly attached to the bottom surface of the culture dish body;
b) Adding a proper amount of cell suspension into the culture dish body, and placing the culture dish body into an incubator for culture;
c) And after the culture is carried out for a preset period of time, observing the cell adherence condition and the cell density, removing the circular cover clamping layer when the cell fusion degree reaches more than 80%, replacing the serum-free culture medium, continuously culturing, and observing the scratch healing condition.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109234143A (en) * | 2018-10-31 | 2019-01-18 | 扬州大学 | A kind of cell scoring devices and its application method |
| CN110229752B (en) * | 2019-06-24 | 2024-03-19 | 上海长海医院 | Micro negative pressure cell culture device |
| CN110387317B (en) * | 2019-08-29 | 2023-06-20 | 贵州大学 | Tieback ware for pathogenicity experiment |
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| JP2005121567A (en) * | 2003-10-20 | 2005-05-12 | Fuji Photo Film Co Ltd | Scratching testing device and scratching test method |
| CN105838607A (en) * | 2016-05-27 | 2016-08-10 | 上海臻和生物科技有限公司 | Cell cultivation insertion component for scratch experiments and application of cell cultivation insertion component |
| CN205974491U (en) * | 2016-08-26 | 2017-02-22 | 夏荣木 | Ration mar board |
| CN107267388A (en) * | 2017-08-08 | 2017-10-20 | 清华大学 | A kind of multi-functional cell co-cultures capsule and its application |
| CN208577725U (en) * | 2018-04-27 | 2019-03-05 | 上海长海医院 | A kind of cell scratch experiment culture dish |
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- 2018-04-27 CN CN201810390218.0A patent/CN108384715B/en active Active
Patent Citations (5)
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| JP2005121567A (en) * | 2003-10-20 | 2005-05-12 | Fuji Photo Film Co Ltd | Scratching testing device and scratching test method |
| CN105838607A (en) * | 2016-05-27 | 2016-08-10 | 上海臻和生物科技有限公司 | Cell cultivation insertion component for scratch experiments and application of cell cultivation insertion component |
| CN205974491U (en) * | 2016-08-26 | 2017-02-22 | 夏荣木 | Ration mar board |
| CN107267388A (en) * | 2017-08-08 | 2017-10-20 | 清华大学 | A kind of multi-functional cell co-cultures capsule and its application |
| CN208577725U (en) * | 2018-04-27 | 2019-03-05 | 上海长海医院 | A kind of cell scratch experiment culture dish |
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