CN117417882A

CN117417882A - Preparation method and equipment of single cell suspension of zebra fish embryo

Info

Publication number: CN117417882A
Application number: CN202311356032.0A
Authority: CN
Inventors: 欧阳刚; 罗大极; 刘飞; 赵媛莉
Original assignee: Institute of Hydrobiology of CAS
Current assignee: Institute of Hydrobiology of CAS
Priority date: 2023-10-18
Filing date: 2023-10-18
Publication date: 2024-01-19
Anticipated expiration: 2043-10-18

Abstract

The invention relates to the technical field of preparation of single cells of zebra fish embryos, in particular to a preparation method and equipment of single cell suspension of zebra fish embryos, wherein the preparation method of the single cell suspension of the zebra fish embryos comprises the steps of collecting fertilized eggs of the zebra fish, developing for 10 hours, and selecting zebra fish eggs in a bud period; stripping egg membranes when the embryo grows to a long pec period, and collecting embryos of which the egg membranes are removed; centrifuging instantaneously to remove embryo culture solution; washing twice with 1ml of pre-chilled PBS, and re-suspending the oomycete membrane removed embryo with 1ml of PBS; transferring 1ml of PBS containing the egg membrane removed embryo to a precooled homogenizer, homogenizing on ice; the single cell suspension was filtered, concentrated and counted. The single cell number can be obtained by using basic equipment to collect eggs, wash, homogenate, filter and count, and the single cell number is used for the ChIP-Seq biological group technology, the operation flow is simple, the cost is low, no impurity is introduced, and the cell yield accuracy is high.

Description

Preparation method and equipment of single cell suspension of zebra fish embryo

Technical Field

The invention relates to the technical field of preparation of single cells of zebra fish embryos, in particular to a preparation method and equipment of single cell suspension of zebra fish embryos.

Background

In recent years, with rapid development of sequencing technology in biomedical research fields, biological technologies such as RNA-Seq, chIP-Seq, ATAC-Seq and the like are increasingly applied in various fields such as medicine, environment, aquatic products and the like. Due to the complexity of the gene expression control mechanism, the integration analysis of a variety of histologic data is increasingly important to explore biological problems from different levels. From the RNA-Seq level we can explore which genes have significant differences, up-or down-regulated; from the ChIP-Seq level, we can study the regulatory role of a particular transcription factor; from the ATAC-Seq we can see that dynamic changes in chromatin accessibility play an important role in transcriptional regulation, since chromatin accessibility is closely related to the binding of regulatory elements or transcription factors.

At present, the comparative hot single-cell sequencing is a novel technology for carrying out high-throughput sequencing analysis on genome, transcriptome and apparent group at the single-cell level, and can reveal the gene structure and the gene expression state of single cells. Thus, preparing a single cell suspension of high quality is a determinant for ensuring sequencing quality. Meanwhile, primary cell culture, establishment of immortalized cell lines, and the like, also require preparation of single cell suspensions. The prior preparation method of the zebra fish embryo single cells has complex steps and high cost.

Disclosure of Invention

The invention aims to provide a preparation method and equipment of a single cell suspension of a zebra fish embryo, which solve the problems of complex preparation steps and high cost of the conventional single cell preparation of the zebra fish embryo.

In order to achieve the above object, in a first aspect, the present invention provides a preparation method of a single cell suspension of zebra fish embryo, comprising the following steps:

(1) Collecting fertilized eggs of zebra fish, developing for 10 hours, and selecting 300 zebra fish eggs in the bud period;

(2) Stripping egg membranes after the development is completed for 48 hours, and collecting 100 embryo pieces of egg membrane removed;

(3) Centrifuging instantaneously to remove embryo culture solution;

(4) Washing twice with 1ml of pre-chilled PBS, and re-suspending the oomycete membrane removed embryo with 1ml of PBS;

specifically, the PBS is gently washed from the top to the embryo, so that the PBS is left from top to bottom, and the situation that the washed PBS has a certain impact force to loosen the periphery of the embryo and cause the falling of the embryo is avoided. After the first wash, the second wash with pre-chilled PBS was continued for 2 minutes.

(5) Transferring 1ml of PBS containing the egg membrane removed embryo to a precooled homogenizer, homogenizing on ice;

(6) Collecting the cell suspension by using a 50ml centrifuge tube, transferring 1ml of the cell suspension to a screen of a filter, and filtering the cell suspension;

(7) Washing the cell filtration in the screen of the filter;

(8) Transferring 1ml of the cell suspension collected by a 50ml centrifuge tube to two 1.5ml EP tubes respectively, centrifuging, and removing the supernatant;

(9) Taking 0.5ml of cell suspension from 50ml centrifuge tubes, adding the cell suspension into two 1.5ml EP tubes in the step (8) respectively, and re-suspending the cells;

(10) The two tube cell suspensions were then combined into one tube and after mixing, the cells were counted.

Wherein, in the step (5), the slurry is homogenized on ice, and the method specifically comprises the following steps:

homogenizing for 5 times up and down, 5 times clockwise, 5 times anticlockwise, and 5 times up and down.

Wherein, the homogenizer in the step (5) is a 5ml glass homogenizer.

Wherein the mesh size in step (6) is 40. Mu.m.

Wherein, the cell filtration in the flushing screen in the step (7) specifically comprises:

the cells in the screen were rinsed with 1ml of PBS and the cell suspension was filtered, and then rinsed with 1ml of PBS to allow the cells in the screen to be sufficiently filtered.

Wherein, the centrifugation speed in the step (8) is 1000rpm/min, and the centrifugation time is 5min.

In a second aspect, the invention also provides a preparation device of the single-cell suspension of the zebra fish embryo, which is applied to the preparation method of the single-cell suspension of the zebra fish embryo in the first aspect, and comprises a transient centrifuge, a glass homogenizer, a micropipette and a full-automatic cell counter;

the instantaneous centrifuge is used for centrifuging embryos with egg membranes removed and removing embryo culture solution;

the glass homogenizer is used for homogenizing the embryo which is washed and resuspended and contains the egg membrane on ice;

the micropipette is used for transferring the homogenized cell suspension to a screen for filtering;

the full-automatic cell counter is used for counting cells after filtration, transfer and re-suspension.

According to the preparation method and the preparation equipment of the zebra fish embryo single-cell suspension, the fertilized eggs of the zebra fish are collected to develop for 10 hours, and zebra fish eggs in the bud period are selected; stripping egg membranes when the embryo grows to a long pec period, and collecting embryos of which the egg membranes are removed; centrifuging instantaneously to remove embryo culture solution; washing twice with 1ml of pre-chilled PBS, and re-suspending the oomycete membrane removed embryo with 1ml of PBS; transferring 1ml of PBS containing the egg membrane removed embryo to a precooled homogenizer, homogenizing on ice; the single cell suspension was filtered, concentrated and counted. The single cell number can be obtained by using basic equipment to collect eggs, wash, homogenate, filter and count, and the single cell number is used for the ChIP-Seq biological group technology, the operation flow is simple, the cost is low, no impurity is introduced, and the cell yield accuracy is high.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a schematic flow chart of a preparation method of a single cell suspension of zebra fish embryos according to an embodiment of the present invention.

FIG. 2 is a TC20 fully automated cell counter (bio-rad) single cell suspension count showing the total single cell suspension cell mass (4.84X106 cells/ml).

FIG. 3 is a photograph of a PAGE gel after library construction.

FIG. 4 is a diagram showing peaks of nucleic acid detection after library construction.

Fig. 5 is a schematic structural view of a filter provided by the present invention.

Fig. 6 is a front view of a filter provided by the present invention.

Fig. 7 is a cross-sectional view taken along line A-A in fig. 6.

In the figure: 101-placing container, 102-first placing piece, 103-annular chute, 104-screen, 105-first inserting portion, 106-second inserting portion, 107-first placing groove, 108-annular supporting table, 109-second placing piece, 110-split box, 111-first liquid inlet boss, 112-first split table, 113-second liquid inlet boss, 114-second split table, 115-liquid outlet hole, 116-third inserting portion, 117-fourth inserting portion, 118-second placing groove, 119-first moving block, 120-second moving block, 121-through groove.

Detailed Description

The following detailed description of embodiments of the invention, examples of which are illustrated in the accompanying drawings and, by way of example, are intended to be illustrative, and not to be construed as limiting, of the invention.

In a first aspect, the present invention provides a method for preparing a single cell suspension of a zebra fish embryo, and specifically, the method for preparing a single cell suspension of a zebra fish embryo may include the following steps:

specifically, zebra fish is AB series and is derived from aquatic organism institute of China academy of sciences. The collecting steps are as follows: the zebra fish is put into a breeding fish tank from the afternoon or evening, and the male zebra fish and the female zebra fish are separated by the baffle plate so as to prevent the male zebra fish and the female zebra fish from mating, and the breeding fish tank is covered at the same time, so that the zebra fish is prevented from jumping out of the fish tank, and the zebra fish are adapted to each other overnight. The next morning, the baffle was removed and ovulation was triggered. When the male zebra fish chases the female zebra fish in the breeding fish tank, the female zebra fish starts to ovulate, the male zebra fish also discharges sperms into water, the eggs are released and fertilized, and the waiting time is about 15-30 minutes. And then the adult zebra fish is moved out of the breeding fish tank by a fishing net. And pouring water and zebra fish eggs into a filter screen to collect the zebra fish eggs, eluting the zebra fish eggs from the filter screen into a culture dish by using filtered fish egg water, and developing for 10 hours, wherein the pH of breeding water is 6.5-7.5, the water hardness is 6-8, and the breeding water temperature is 25-26 ℃. Selecting zebra fish eggs in the bud period, wherein the zebra fish eggs in the bud period are in one stage of the embryo development process, at the moment, egg cells start to divide and differentiate to form an embryo structure of fish, the zebra fish eggs in the bud period have the characteristics of cell division, placenta formation, embryo differentiation, yolk sac development and embryo suspension, the subsequent egg membrane stripping is convenient, embryos are collected, namely the cells of the zebra fish eggs start to divide mitotically, and the divided cells form an embryo with a plurality of cells; in embryonic cells, one cell develops into a blastoderm, one of the most important cells in an embryo, which is capable of differentiating into various organs and tissues in the future; as the blastoderm develops, the embryo begins to differentiate into various organs and tissues, such as the eye, brain, heart, muscle, bone, etc.; during the development process of zebra fish eggs, the yolk sac also gradually develops; the yolk sac is an important source for supplying nutrition to the embryo, and gradually increases as the embryo grows continuously; during development, the embryo will gradually take up nutrients in the yolk sac and begin to float in the water.

specifically, 48 hours is a long pec period, a pair of zebra fish spawns is about 200-500 and 300 are common, and the 48 hours zebra fish embryo is selected in the invention, because earlier studies show that the 48 hours embryo can be used for observing wild zebra fish embryo and mutant zebra fish embryo, and the phenomenon that the red blood cells of the mutant zebra fish embryo are reduced can be found, so that a required cell sample can be provided for the ChP_seq. Meanwhile, when early cell research is needed, the early cell research is generally needed to be divided into three groups, three repeated samples are made, and the number of embryos is enough; when the cell research is carried out in earlier stage, more fish eggs are needed, the number of eggs laid by a pair of fishes is insufficient, at the moment, a plurality of pairs of fishes are needed to lay eggs, and a certain difference exists in the egg laying genetic background of the plurality of pairs of fishes. Meanwhile, the zebra fish embryo in the long pec period is basically a complete juvenile fish, has all structures, and basically has all cell types, and is different from the early embryo adopted in the prior art, the cell dispersion degree is insufficient due to the existence of some connective tissues, so that experimental deviation is caused finally because of the cell number problem. After 48 hours of development, zebra fish eggs are taken out and placed on a glass slide, egg membranes are gently peeled off by forceps, the eggs are placed under an dissecting mirror for observation, and the development process is recorded according to the marked marks and development period on a sampling tube. The forceps were ST-16 (model number) of VETUS (brand).

(3) Centrifuging instantaneously to remove embryo culture solution;

specifically, the culture dish was placed in a transient centrifuge, the transient centrifuge was started, the centrifugation rate was set to 1000rpm/min, and the centrifugation time was 5min, to remove the embryo culture fluid. The instantaneous centrifuge is a S1010E (model) palm centrifuge of the scilex scilodex (brand).

specifically, the PBS is phosphate buffer solution, and the pre-cooled PBS can better maintain the activity and stability of cells, avoid the occurrence of cell membrane rupture, cell degeneration and other conditions caused by the overhigh external temperature, and ensure the accuracy and reliability of experimental results. The first cleaning can remove residues of the culture medium and cell fragments generated in the culture process, so that the purity and activity of cells are improved, the second cleaning can more thoroughly remove bacteria and other pollutants possibly existing, the purity of the cells is improved, the cell pollution is reduced, and the cell damage caused by improper operation or too high concentration of reagents can be avoided when the cleaning is carried out twice, so that the experimental result is influenced. After the two times of cleaning, the embryo from which the egg membrane is removed is resuspended by 1ml of PBS, so that the microenvironment of the cells can be maintained, the activity of the cells can be maintained, and the subsequent experimental operation can be facilitated. PBS is Solarbio (brand), P1022 (cat number).

specifically, a precooled homogenizer and a centrifuge tube are prepared, 1ml of PBS containing embryos with egg removal membranes is placed in the centrifuge tube, the centrifuge tube is sealed by a sealing membrane, the centrifuge tube is placed in an instantaneous centrifuge, the instantaneous centrifuge is started, the centrifugation speed is set to 1000rpm/min, the centrifugation time is 5min, homogenization on ice is carried out for 5 times in a clockwise direction for 5 times, homogenization on ice is carried out for 5 times in a anticlockwise direction for 5 times, and then homogenization on ice is carried out for 5 times in a anticlockwise direction for splitting cells into smaller fragments. The homogenizer is a 5ml glass homogenizer, and the glass homogenizer can effectively grind the tissue sample into uniform fine particles, so that the uniformity of the sample is improved, and the accuracy and the reliability of subsequent experiments are ensured. Meanwhile, the glass is an inert material and does not react with the tissue sample, so that the pollution of the sample can be reduced to the greatest extent by using the glass homogenizer, and the reliability of the experimental result is improved. In addition, the glass homogenizer is easy to clean and maintain, and the use cost is reduced.

specifically, the screen mesh aperture is 40 mu m, can filter great impurity and dead cell, can not hinder the passage of target cell simultaneously, and be difficult for causing the jam when filtering, can guarantee the accuracy of filtration quality and experimental result. The screen is biosharp (brand), BS-40-CS (model). The cell suspension was transferred using a micropipette, which was Eppendorf (brand), 5425 (model), germany.

(7) Washing the cell filtration in the screen of the filter;

specifically, 1ml of PBS was added to wash the cells in the screen, the cell suspension was filtered, and 1ml of PBS was added to wash the cells in the screen, so that the cells in the screen were sufficiently filtered. The two flushes have the following effects: removing impurities and dead cells in the cell suspension, and improving the purity of the cells; cleaning cells, removing impurities and harmful substances on the surfaces of the cells, so that the cells are purer and healthier; the pH balance and osmotic pressure of the culture solution are reestablished, which is beneficial to the growth and division of cells; the cell concentration is adjusted to provide a suitable cell density for subsequent experiments or cultures. Referring to fig. 5 to 7, fig. 5 is a schematic structural diagram of a filter according to the present invention. Fig. 6 is a front view of a filter provided by the present invention. Fig. 7 is a cross-sectional view taken along line A-A in fig. 6. The filter comprises a holding vessel 101, a first holding member 102 and a diverter assembly; the placement container 101 has an annular chute 103, the first placement member 102 is inserted into the annular chute 103 and the placement container 101, and the screen 104 is detachably connected to the first placement member 102 and is located in the placement container 101. The upper end of the placement container 101 is provided with a first opening, the end face of the placement container is provided with an annular chute 103 with a certain depth, the first placement member 102 is an annular body and is provided with a first insertion part 105 and a second insertion part 106, a first placement groove 107 is formed in the middle of the first insertion part 105 and the second insertion part 106, the placement container 101 is inserted into the annular chute 103 through the first insertion part 105 of the first placement member 102, the first placement groove 107 accommodates the inner wall of the placement container 101, the screen 104 is fixed, screens 104 with different specifications can be replaced according to experimental requirements, rapid placement is facilitated, and the efficiency is improved.

The diversion assembly comprises an annular supporting table 108, a second placing piece 109, a diversion box 110, a first liquid inlet boss 111, a first diversion table 112, a second liquid inlet boss 113 and a second diversion table 114; the annular supporting table 108 and the first placing member 102 are integrally formed and located at one side of the placing container 101, which is close to the screen 104, the second placing member 109 is inserted into the annular chute 103 and is propped against the annular supporting table 108, the split box 110 is fixedly connected with the second placing member 109 and located in the placing container 101, a liquid outlet hole 115 is formed in the bottom of the split box, the first liquid inlet boss 111 is fixedly connected to the split box 110 and communicated with the split box 110, the first split box 112 is fixedly connected to the split box 110, the second liquid inlet boss 113 is fixedly connected to the first split box 112, one end of the second split box 114 is located in the first liquid inlet boss 111, and the second split box 114 is located in the first split box 112. The second placing member 109 has a third inserting portion 116 and a fourth inserting portion 117, a second placing groove 118 is formed between the third inserting portion 116 and the fourth inserting portion 117, the second placing groove 118 accommodates the first placing member 102, the third inserting portion 116 is inserted into the annular chute 103 and is in contact with the first inserting portion 105, the fourth inserting portion 117 is inserted into the placing container 101 and is in contact with the second inserting portion 106, and is located on the annular supporting table 108, and the split box 110 is fixed on the fourth inserting portion 117. The split box 110 is a cylinder with a liquid inlet hole at the upper part and a hollow inside, the first liquid inlet boss 111 and the second liquid inlet boss 113 are cylinders with central holes, the first split table 112 and the second split table 114 are provided with second openings at the upper and lower parts, the inside is hollow, the cross section is trapezoidal, the split box is provided with an upper bottom edge, a lower bottom edge and two bevel edges, the length of the upper bottom edge is smaller than that of the lower bottom edge, PBS for flushing cells is poured from the holes of the first liquid inlet boss 111, a part of PBS is guided to the inclined surfaces of the first split table 112 by the inner wall of the first liquid inlet boss 111, and then a plurality of liquid outlet holes 115 flowing to the outer side of the split box 110 drop to flush the outer side of the cells; the other part flows from the center of the first liquid inlet boss 111 to the second liquid inlet boss 113, and the part of the PBS entering the second liquid inlet boss 113 directly flows into the second flow dividing table 114 to flow to the center of the flow dividing box 110, flows out from the liquid outlet hole 115 to wash the center of the cell, enters the space between the first flow dividing table 112 and the second flow dividing table 114, flows into the space between the first flow dividing table 112 and the second flow dividing table 114 from the inclined plane of the second flow dividing table 114, flows out from the liquid outlet hole 115 to wash the center of the cell and the position between the edges, so that the PBS is divided to different positions, the cells are sufficiently washed, the washing effect is improved, the impact force of the PBS after drainage is not large, and the impact influence on the cells is not dispersed.

The filter further comprises a first moving block 119 and a second moving block 120, the first moving block 119 is fixedly connected with the first placing member 102, and penetrates through the second placing member 109 to be located outside the placing container 101, and the second moving block 120 is fixedly connected to the second placing member 109. The second placement member 109 has a through slot 121, and the first moving block 119 is positioned to be partially inserted into the through slot 121, and the first moving block 119 is configured to be easily removed and moved into the screen 104. The second moving block 120 is provided to facilitate the taking and moving in and out of the split box 110.

The specific flow is as follows: a screen 104 having a pore diameter of 40 μm is selected to be fixed to the second insertion portion 106, the first insertion portion 105 of the first placement member 102 is inserted into the annular chute 103, the second insertion portion 106 is inserted into the placement container 101, and the inner wall of the placement container 101 is positioned in the first placement groove 107, so that the screen 104 is positioned in the placement container 101. The third insertion portion 116 of the second placement element 109 is inserted into the annular chute 103, the second moving block 120 is inserted into the through groove 121, the fourth insertion portion 117 is inserted into the placement container 101 and is located on the annular supporting table 108, the split box 110 is fixed, 1ml of pbs is poured into the first liquid inlet boss 111, and the outside, the center, the outer side and the center of the cell are flushed by the split box 110, so that the cell suspension is filtered, and the flushing effect is improved. And then 1ml of PBS is poured into the first liquid inlet boss 111, and is drained through the shunt box 110 to flush the outer side, the center, the outer side and the center of the cell, so that the flushing effect is improved.

in particular, the EP tube is an enzyme-free EP tube, which can well preserve various bioactive substances in the cell sample, as well as the structure and function of the cells, and maintain the original state of the cell sample. In addition, the enzyme-free EP tube can be used for preserving a cell sample at a low temperature, prolonging the shelf life of the cell sample, and has high light transmittance, thereby being beneficial to cell observation and detection. Is Servicebio (brand), EP-150-M (product number). After transfer, the centrifugation speed was 1000rpm/min and the centrifugation time was 5min, and after centrifugation, the centrifuge tube was gently turned over to separate the cell pellet and supernatant. The supernatant was carefully aspirated with a micropipette.

Specifically, the full-automatic cytometer is bio-rad (brand) in the United states, TC20 (model). The sample was added to the blue-pan dye, blown up and down, gently mixed, and the sample mixture was added to the cell port on one side of the cell counter plate. Optical parameters are used to distinguish between different types or individuals. In the measuring process, a special light source irradiates the sample to generate diffuse light, so that different types or individuals in the sample are separated and counted through detecting different optical characteristics such as absorbed light, scattered light or fluorescence. Referring to FIG. 2, FIG. 2 shows the results of a TC20 fully automated cell counter (bio-rad) single cell suspension count showing the total single cell suspension cell mass (4.84X10) ⁶ cells/ml)。

Referring to fig. 1, fig. 1 is a schematic flow chart of a preparation method of a single cell suspension of zebra fish embryo according to an embodiment of the invention. According to the preparation method of the zebra fish embryo single-cell suspension, fertilized eggs are collected, embryo egg membranes are normalized in development period, embryo egg membrane removal is carried out by stripping, embryo egg membrane removal is carried out by cleaning, embryo egg membrane removal is carried out by homogenizing, single-cell suspension is filtered, single-cell suspension is concentrated, single-cell suspension is counted, the number of single cells can be obtained by only using basic equipment, cleaning, homogenizing, filtering and counting, the operation flow is simple, no impurities are introduced, the cell yield accuracy is high, the cells are separated based on adhesion breaking effect by adopting a chemical method in the prior art, salt ions are introduced, influence is generated on intracellular gene signal transmission to a certain extent, false positive results are caused when single-cell sequencing is carried out in the later period, meanwhile, more operation steps are needed for removing the introduced salt ions, the operation flow becomes complicated and complicated, enzymes are not used, enzyme impurities are not introduced, the time required for enzymolysis reaction is long, and the method in the prior art is expensive, a series of solutions are required to be manpower, time and other reagent cost are required. Referring to fig. 3, fig. 3 is a PAGE gel diagram after library construction. The 250-500bp library was excised, recovered with gel and sequenced. The data from sequencing can be used to analyze whether the transcription factor binds to the promoter region or the gene body of the gene, and whether the gene is functionally linked by GO analysis. Referring to FIG. 4, FIG. 4 is a peak diagram of nucleic acid detection after library construction. The single cell obtained by the preparation method of the zebra fish embryo single cell suspension can meet the requirements of ChIP-Seq experiment. The invention can be used for preparing tissue single cell suspension rapidly and simply, is beneficial to establishing and optimizing an in-vitro culture technology of adult tissue cells, provides important reference data for establishing a cell line, has important theoretical and application significance in the research fields of gene functions, genetic breeding and the like, and has low cost.

In another embodiment, after 48 hours of development, the method further comprises, before stripping the egg membrane: shooting the developed zebra fish eggs based on a microscope to obtain the number of dead eggs and the number of normal eggs;

after the egg membrane is stripped and 100 embryos from the egg membrane are collected, the method further comprises: shooting embryos with egg membranes removed based on a microscope to obtain the number of complete embryos and the number of incomplete embryos;

the two tube cell suspensions are then combined into one tube, and after mixing, the method further comprises, after cell counting: and obtaining the cell count number, the dead egg number, the normal egg number, the complete embryo number and the incomplete embryo number for visual display. And the development states of the zebra fish in different time periods are counted, analysis and comparison are carried out, subsequent experimental verification is facilitated, and an accurate conclusion is obtained.

In this embodiment, the specific implementation details refer to the specific implementation details of the first aspect, and are not repeated herein.

The foregoing disclosure is only illustrative of one or more preferred embodiments of the present application and is not intended to limit the scope of the claims hereof, as it is to be understood by those skilled in the art that all or part of the process of implementing the described embodiment may be practiced otherwise than as specifically described and illustrated by the appended claims.

Claims

1. The preparation method of the single cell suspension of the zebra fish embryo is characterized by comprising the following steps:

(3) Centrifuging instantaneously to remove embryo culture solution;

(7) Washing the cell filtration in the screen of the filter;

2. The method of claim 1, wherein the step (5) comprises homogenizing on ice:

3. The method of claim 1, wherein the homogenizer in step (5) is a 5ml glass homogenizer.

4. The method of claim 1, wherein the mesh size in step (6) is 40 μm.

5. The method of claim 1, wherein the step (7) of washing in-screen cell filtration comprises:

6. The method of claim 1, wherein the centrifugation rate in step (8) is 1000rpm/min and the centrifugation time is 5min.

7. A preparation device of a single cell suspension of zebra fish embryos, which is applied to the preparation method of the single cell suspension of zebra fish embryos according to any one of claims 1 to 6, and is characterized by comprising a transient centrifuge, a glass homogenizer, a micropipette and a full-automatic cell counter;