CN111378613B - Amphibious animal cell dissociation kit - Google Patents
Amphibious animal cell dissociation kit Download PDFInfo
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- CN111378613B CN111378613B CN201811609987.1A CN201811609987A CN111378613B CN 111378613 B CN111378613 B CN 111378613B CN 201811609987 A CN201811609987 A CN 201811609987A CN 111378613 B CN111378613 B CN 111378613B
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- 238000010494 dissociation reaction Methods 0.000 title abstract description 12
- 230000005593 dissociations Effects 0.000 title abstract description 12
- 210000004102 animal cell Anatomy 0.000 title abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 155
- 210000001519 tissue Anatomy 0.000 claims abstract description 48
- YRQNKMKHABXEJZ-UVQQGXFZSA-N chembl176323 Chemical compound C1C[C@]2(C)[C@@]3(C)CC(N=C4C[C@]5(C)CCC6[C@]7(C)CC[C@@H]([C@]7(CC[C@]6(C)[C@@]5(C)CC4=N4)C)CCCCCCCC)=C4C[C@]3(C)CCC2[C@]2(C)CC[C@H](CCCCCCCC)[C@]21C YRQNKMKHABXEJZ-UVQQGXFZSA-N 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 21
- 239000002738 chelating agent Substances 0.000 claims abstract description 17
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 17
- 210000003205 muscle Anatomy 0.000 claims abstract description 17
- 102000004142 Trypsin Human genes 0.000 claims abstract description 14
- 108090000631 Trypsin Proteins 0.000 claims abstract description 14
- 108010007093 dispase Proteins 0.000 claims abstract description 14
- 239000012588 trypsin Substances 0.000 claims abstract description 14
- 102000029816 Collagenase Human genes 0.000 claims abstract description 12
- 108060005980 Collagenase Proteins 0.000 claims abstract description 12
- 229960002424 collagenase Drugs 0.000 claims abstract description 12
- 238000010008 shearing Methods 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims description 14
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- 241000269333 Caudata Species 0.000 claims description 11
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 10
- 238000002791 soaking Methods 0.000 claims description 9
- RDEIXVOBVLKYNT-VQBXQJRRSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(1-aminoethyl)oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;(2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(aminomethyl)oxan-2-yl]o Chemical compound OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@@H](CN)O2)N)[C@@H](N)C[C@H]1N.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@H](O2)C(C)N)N)[C@@H](N)C[C@H]1N.O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N RDEIXVOBVLKYNT-VQBXQJRRSA-N 0.000 claims description 5
- 229930182555 Penicillin Natural products 0.000 claims description 5
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 5
- 229940049954 penicillin Drugs 0.000 claims description 5
- 229960005322 streptomycin Drugs 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 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 description 4
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 claims description 4
- 210000004027 cell Anatomy 0.000 abstract description 62
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- 210000002966 serum Anatomy 0.000 description 18
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- 239000002609 medium Substances 0.000 description 8
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- 229940088598 enzyme Drugs 0.000 description 7
- 239000012528 membrane Substances 0.000 description 6
- 238000002156 mixing Methods 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000030833 cell death Effects 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 230000003204 osmotic effect Effects 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 229930182566 Gentamicin Natural products 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 238000003320 cell separation method Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000003414 extremity Anatomy 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 210000001364 upper extremity Anatomy 0.000 description 2
- RDEIXVOBVLKYNT-HDZPSJEVSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-[(1r)-1-aminoethyl]oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;(2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(aminomethyl)oxan-2 Chemical compound OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@@H](CN)O2)N)[C@@H](N)C[C@H]1N.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@H](O2)[C@@H](C)N)N)[C@@H](N)C[C@H]1N.O1[C@H]([C@@H](C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N RDEIXVOBVLKYNT-HDZPSJEVSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000219764 Dolichos Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 241000187398 Streptomyces lividans Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an amphibian animal cell dissociation kit. The kit of the invention comprises the following reagents: the reagent C contains collagenase I and dispase II, and the concentration is 43000-53000U/100ml, 90-110units/100ml in sequence; reagent D contains collagenase I at a concentration of 55000-65000U/100ml. Reagent E contains trypsin and metal ion chelating agent, and the concentration is 120000-180000USPU/100ml, 0.03-0.07g/100ml. Reagent F contains collagenase II at a concentration of 50000-55000U/100ml. The invention also provides a method for dissociating the tissue of the amphibian to obtain single cells, which comprises the following steps: shearing skin tissue, and treating with reagent C; shearing muscle tissues, adding the reagent D for treatment, and then adding the reagent E for treatment; skeletal tissue, minced, treated with reagent F. The invention can be applied to single-cell research of limb skin, muscle and skeletal tissue of various amphibians.
Description
Technical Field
The invention relates to an amphibian cell dissociation kit.
Background
Many amphibians represented by salamanders have strong regeneration capability, but the cell separation and culture methods are to be broken through due to the great differences between the characteristics of the amphibians, such as cell osmotic pressure and life adaptation temperature, and mammals.
At present, most laboratories taking amphibians as experimental objects take tissue integrity as research objects, lack of exploration of cell heterogeneity and remain in existence in some scientific problems. The most common method for the mammal to dissociate the tissue or the cultured cells into single cells is enzymolysis, and pancreatin is the most commonly used enzyme in the laboratory because of low price and strong effect.
The problems in the prior art are as follows:
(1) If the dissociation method of each type of mammalian cells is adopted, the dissociation temperature is not suitable, the enzyme activity cannot be controlled, the cells are difficult to maintain in the same state as in vivo, and if the dissociation temperature is taken as a research object, the authenticity of the experimental result cannot be ensured;
(2) The dissociation efficiency is low, more tissues need to be dissociated in order to meet the research requirement, and the experiment cost is increased;
(3) The pancreatin has strong effect, is easy to cause cell membrane damage and apoptosis, is difficult to ensure the cell activity rate, and is unfavorable for subsequent research.
Disclosure of Invention
The invention aims to provide an amphibian cell dissociation kit.
The invention claims a kit for dissociating amphibian tissue to obtain cells, the kit comprising the following reagents: reagent C, reagent D, reagent E and reagent F.
The reagent C contains collagenase I and dispase II, wherein the concentration of the collagenase I is 43000-53000U/100ml, and the concentration of the dispase II is 90-110units/100ml.
The reagent D contains collagenase I, and the concentration of the collagenase I is 55000-65000U/100ml.
The reagent E contains trypsin and metal ion chelating agent, wherein the concentration of the trypsin is 120000-180000USP U/100ml, and the concentration of the metal ion chelating agent is 0.03-0.07g/100ml.
The reagent F contains collagenase II, and the concentration of the collagenase II is 50000-55000U/100ml.
The reagent C contains collagenase I and dispase II, wherein the concentration of the collagenase I is 48000U/100ml, and the concentration of the dispase II is 100units/100ml.
The reagent D contains collagenase I, and the concentration of the collagenase I is 60000U/100ml.
The reagent E contains trypsin and a metal ion chelating agent, wherein the concentration of the trypsin is 150000USP U/100ml, and the concentration of the metal ion chelating agent is 0.03-0.07g/100ml.
Reagent F contains collagenase II with a concentration of 52200U/100ml.
The reagent C contains collagenase I and dispase II, wherein the concentration of the collagenase I is 0.1-0.3g/100ml, and the concentration of the dispase II is 0.1-0.3g/100ml.
The reagent D contains collagenase I, and the concentration of the collagenase I is 0.2-0.3g/100ml.
The reagent E contains trypsin and metal ion chelating agent, wherein the concentration of the trypsin is 0.4-0.6g/100ml, and the concentration of the metal ion chelating agent is 0.03-0.07g/100ml.
The reagent F contains collagenase II with the concentration of 0.1-0.3g/100ml.
The reagent C contains collagenase I and dispase II, wherein the concentration of the collagenase I is 0.2g/100ml, and the concentration of the dispase II is 0.2g/100ml.
The reagent D contains collagenase I, and the concentration of the collagenase I is 0.25g/100ml.
The reagent E contains trypsin and a metal ion chelating agent, wherein the concentration of the trypsin is 0.5g/100ml, and the concentration of the metal ion chelating agent is 0.05g/100ml.
Reagent F contains collagenase II at a concentration of 0.2g/100ml.
The kit can be used for dissociating from skin, muscle and bone of amphibians to obtain cells.
The invention also protects the application of the kit, and cells are obtained for dissociating organs or tissues of the amphibian.
The invention also provides a kit for dissociating skin tissues of amphibians to obtain cells, comprising the reagent C. The invention also protects the application of the kit, and cells are obtained for dissociating skin tissues of the amphibian.
The invention also provides a kit for dissociating amphibian muscle tissue to obtain cells, comprising the reagent D and the reagent E. The invention also protects the application of the kit, and cells are obtained for dissociating the muscle tissues of the amphibians.
The invention also provides a kit for dissociating bone tissue of an amphibian to obtain cells, comprising the reagent F. The invention also protects the application of the kit, and cells are obtained for dissociating bone tissues of the amphibian.
The kit of any of the above may further comprise reagent a and/or reagent B.
Reagent A is a cleaning reagent.
Reagent B is a pretreatment reagent.
The reagent B contains 0.05-0.15g/100ml metal ion chelating agent.
The reagent B specifically contains 0.1g/100ml of metal ion chelating agent.
Any of the above metal ion chelating agents may specifically be EDTA-Na 2 。EDTA-Na 2 In particular, it can be provided in pure form or as a hydrated compound.
Any of the above reagents C may be composed of collagenase I, dispase II and reagent A.
Any of the above reagents D may consist of collagenase I and reagent A.
Any of the reagents E above may consist of a reagent a containing trypsin, a metal ion chelator.
Any of the above reagents F may consist of collagenase II and reagent A.
Any of the above reagents A contained 3-4mg/100ml gentamicin sulfate, 80-120 units/ml penicillin (base) and 80-120. Mu.g/ml streptomycin (base).
Any of the above reagents A contained 3.5mg/100ml gentamicin sulfate, 100 units/ml penicillin (base) and 100. Mu.g/ml streptomycin (base).
The reagent A consists of gentamicin sulfate, penicillin (alkali), streptomycin (alkali), DPBS and enzyme-free water.
The preparation method of the reagent A specifically comprises the steps of taking all raw materials according to the table 2, fully dissolving and uniformly mixing, filtering with a 0.22 mu m filter membrane, and collecting filtrate to obtain the reagent A.
The invention also provides a method for dissociating the tissue of the amphibian to obtain single cells, which comprises the following steps:
taking skin tissue, shearing the skin tissue, and treating the skin tissue with the reagent C;
shearing muscle tissues, adding the reagent D for treatment, and then adding the reagent E for treatment;
bone tissue was taken, minced and treated with the reagent F.
The method for obtaining single cells by dissociating the tissue of the amphibian specifically comprises the following steps:
taking skin tissue, shearing, placing in a reagent C, soaking for 1-3h at room temperature, adding 2 times of alpha-MEM culture medium containing 10% of serum to stop digestion, filtering with a 70 μm cell filter screen, collecting filtrate, centrifuging at 1000rpm for 5min, collecting cell precipitate, and re-suspending with alpha-MEM culture medium containing 10% of serum;
taking muscle tissue, shearing, placing in a reagent D, soaking for 1-2h at room temperature, adding an equal volume of the reagent E, continuously soaking for 20min at room temperature, adding 2 times of alpha-MEM culture medium containing 10% of serum to stop digestion, filtering with a 70 mu m cell filter screen, collecting filtrate, centrifuging at 1000rpm for 5min, collecting cell precipitate, and re-suspending with the alpha-MEM culture medium containing 10% of serum;
the bone tissue was taken, washed three times (3 min each) in reagent A, and then minced to about 1mm with a scalpel 3 Is then placed in reagent F, immersed for 1.5-2h at room temperature (at which time the tissue mass is substantially disappeared and the solution becomes turbid), then 2 volumes of a-MEM medium containing 10% serum are added to terminate digestion, then the filtrate is filtered and collected with a 70 μm cell filter, centrifuged at 1000rpm for 5min, the cell pellet is collected, and resuspended in a-MEM medium containing 10% serum.
The invention also provides a method for obtaining cells by dissociating skin tissues of amphibians, which comprises the following steps: skin tissue was taken, minced, and treated with the reagent C. The method specifically comprises the following steps: taking skin tissue, shearing, placing in a reagent C, soaking for 1-3h at room temperature, adding 2 times of alpha-MEM culture medium containing 10% of serum to stop digestion, filtering with a 70 μm cell filter screen, collecting filtrate, centrifuging at 1000rpm for 5min, collecting cell precipitate, and re-suspending with alpha-MEM culture medium containing 10% of serum;
the invention also provides a method for dissociating the muscle tissue of the amphibian to obtain cells, which comprises the following steps: and taking muscle tissues, shearing the muscle tissues, adding the reagent D for treatment, and then adding the reagent E for treatment. The method specifically comprises the following steps: taking muscle tissue, shearing, placing in a reagent D, soaking for 1-2h at room temperature, adding an equal volume of the reagent E, continuously soaking for 20min at room temperature, adding 2 times of alpha-MEM culture medium containing 10% of serum to stop digestion, filtering with a 70 mu m cell filter screen, collecting filtrate, centrifuging at 1000rpm for 5min, collecting cell precipitate, and re-suspending with the alpha-MEM culture medium containing 10% of serum;
the invention also provides a method for obtaining cells by dissociating bone tissue of the amphibian, which comprises the following steps: bone tissue was taken, minced and treated with the reagent F. The method specifically comprises the following steps: the bone tissue was taken, washed three times (3 min each) in reagent A, and then minced to about 1mm with a scalpel 3 Is then placed in reagent F, immersed for 1.5-2h at room temperature (at which time the tissue mass is substantially disappeared and the solution becomes turbid), then 2 volumes of a-MEM medium containing 10% serum are added to terminate digestion, then the filtrate is filtered and collected with a 70 μm cell filter, centrifuged at 1000rpm for 5min, the cell pellet is collected, and resuspended in a-MEM medium containing 10% serum.
The method of any one of the above further comprises the following washing and pretreatment steps: the organs are taken, washed in 70% ethanol for 10min, then washed in reagent A for three times (3 min each time), the attachments on the tissue surfaces are fully removed, and then the organs are placed in reagent B and soaked for 10-15min at room temperature.
The invention provides a kit for dissociation of tissue of an amphibian to obtain single cells, and further provides a method for obtaining the single cells of the amphibian by using the kit.
In the kit provided by the invention, the cleaning reagent, the pretreatment reagent and the enzyme dissolving solution with specific formulas are mild in reagent property, so that damage to cells caused by excessive osmotic pressure can be avoided, and tissue dissociation of the amphibian can be rapidly and effectively completed to obtain single cells. The method provided by the invention utilizes the mechanical action, enzymolysis action and centrifugal force action to dissociate cells. The invention establishes a simple, stable and efficient single-cell separation method for the amphibian, which can not dissociate the amphibian before dissociating, and expands the species range of single-cell level research.
At present, most of the research on amphibians in laboratories stays at the tissue level, and due to the specificity of amphibian species (cell osmotic pressure and proper cell culture temperature are lower than those of mammals), the enzymolysis method of related tissues of the mammals is adopted, so that the problems of low dissociation efficiency, great damage to cells, inconvenience to subsequent research and the like exist. The invention can be simultaneously applied to amphibians in different development stages, and can be applied to single-cell research of limb skin, muscle and skeletal tissues of various amphibians.
Drawings
Fig. 1 is a single cell picture (100X) of the muscle tissue of the salamander in mexico.
Fig. 2 is a picture (100X) of single cells of the skin tissue of the salamander in mexico.
Fig. 3 is a picture (100X) of single cells of bone tissue of a salamander of mexico.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
The individual starting materials used in the examples are shown in Table 1.
TABLE 1
The preparation method of 1000 x gentamicin solution comprises the following steps: 350mg of gentamicin sulphate (BBI, A620217-5G, powder) were dissolved in water and taken up to a volume of 10ml with water.
DPBS is a phosphate buffer solution of dolichos and the common standard PBS is free of calcium and magnesium ions.
Example 1 preparation of kit
The kit consists of the following components: reagent A, reagent B, reagent C, reagent D, reagent E and reagent F.
Reagent A is a cleaning reagent. The preparation method of the reagent A comprises the following steps: the raw materials are taken according to the table 2, fully dissolved and uniformly mixed, then filtered by a 0.22 mu m filter membrane, and the filtrate is collected, thus obtaining the reagent A.
Table 2 (total volume 125 ml)
| Raw materials | Dosage of |
| DPBS | 100ml |
| 1000 x gentamicin | 0.125ml |
| 100x Streptomyces lividans | 1.25ml |
| Enzyme-free water | 23.625ml |
Reagent B is a pretreatment reagent. The preparation method of the reagent B comprises the following steps: EDTA-Na 2 -2H 2 O is added into the reagent A, heated to be dissolved, then the pH value is adjusted to 7.0 by sodium hydroxide, and then the reagent A is used for volume fixation to 100ml, namely the reagent B. In reagent B, the concentration of EDTA was 0.1g/100ml.
Reagent C is enzyme solution. The preparation method of the reagent C comprises the following steps: adding 0.5g of collagenase I and 0.5g of dispase II into 25ml of reagent A, fully dissolving and uniformly mixing, filtering with a 0.22 mu m filter membrane, and collecting filtrate, namely a storage solution; 1ml of the stock solution was mixed with 9ml of reagent A to obtain reagent C. In reagent C, the concentration of collagenase I was 0.2g/100ml (corresponding to 48000U/100 ml), and the concentration of dispase II was 0.2g/100ml (corresponding to 100units/100 ml).
Reagent D is enzyme solution. The preparation method of the reagent D comprises the following steps: adding 0.5g collagenase I into 25ml reagent A, fully dissolving and uniformly mixing, filtering with a 0.22 mu m filter membrane, and collecting filtrate, namely a storage solution; 1.25ml of stock solution was mixed with 8.75ml of reagent A to give reagent D. In reagent D, the concentration of collagenase I was 0.25g/100ml (corresponding to 60000U/100 ml).
Reagent E is enzyme solution. The preparation method of the reagent E comprises the following steps: trypsin and EDTA-Na-2H 2 Adding O into 25ml of reagent A, fully dissolving and uniformly mixing, filtering with a 0.22 mu m filter membrane, and collecting filtrate to obtain a storage solution; 1ml of the stock solution was mixed with 9ml of reagent A to obtain reagent E. In reagent E, the concentration of trypsin was 0.5g/100ml (corresponding to 150000USP U/100 ml), EDTA-Na 2 -2H 2 The concentration of O was 0.05g/100ml.
Reagent F is enzyme solution. The preparation method of the reagent F comprises the following steps: adding 0.5g collagenase II into 25ml reagent A, fully dissolving and uniformly mixing, filtering with a 0.22 mu m filter membrane, and collecting filtrate, namely a storage solution; 1ml of the stock solution was mixed with 9ml of reagent A to obtain reagent F. In reagent F, the concentration of collagenase II was 0.2g/100ml (corresponding to 52200U/100 ml).
Example 2 method of Using the kit
1. Samples (organs or tissues of amphibians, such as one of the limbs) were taken, washed in 70% ethanol for 10min, then three times (3 min each time) in reagent a, and the attachments on the tissue surface were removed thoroughly.
2. And (3) taking the tissue subjected to the step (1), placing the tissue in a reagent B, soaking the tissue for 10-15min at room temperature, and then separating skin tissue.
3. Taking the skin tissue obtained in the step 2, shearing, placing in a reagent C, soaking for 1-3h at room temperature, adding 2 times of alpha-MEM culture medium containing 10% of serum to stop digestion, filtering with a 70 μm cell filter screen, collecting filtrate, centrifuging at 1000rpm for 5min, collecting cell precipitate, and re-suspending with the alpha-MEM culture medium containing 10% of serum.
4. After the completion of step 2, muscle tissue was peeled from the remaining tissue, sheared, placed in reagent D, immersed for 1-2 hours at room temperature, then an equal volume of reagent E was added and immersed for 20 minutes at room temperature, then 2 volumes of 10% serum-containing alpha-MEM medium were added to terminate digestion, then the filtrate was filtered and collected with a 70 μm cell strainer, centrifuged at 1000rpm for 5 minutes, cell pellet was collected, and resuspended with 10% serum-containing alpha-MEM medium.
5. After completion of step 2, bone tissue was peeled from the remaining tissue, washed three times (3 min each) in reagent A, and then minced to about 1mm with a scalpel 3 Is then placed in reagent F, immersed for 1.5-2h at room temperature (at which time the tissue mass is substantially disappeared and the solution becomes turbid), then 2 volumes of a-MEM medium containing 10% serum are added to terminate digestion, then the filtrate is filtered and collected with a 70 μm cell filter, centrifuged at 1000rpm for 5min, the cell pellet is collected, and resuspended in a-MEM medium containing 10% serum.
Example 3 use of the kit
The salamander of mexico takes the right upper limb as a sample. The procedure is as in example 2.
Fig. 1 is a single cell picture (100X) of the muscle tissue of the salamander in mexico.
Fig. 2 is a picture (100X) of single cells of the skin tissue of the salamander in mexico.
Fig. 3 is a picture (100X) of single cells of bone tissue of a salamander of mexico.
The result shows that the kit provided by the invention can effectively dissociate the tissues of the amphibian (such as salamander), and the cell survival rate is more than 85% (the cell survival rate is the proportion of the number of the survival cells counted by observation under a microscope to the total number of the cells).
Comparative example,
The salamander of mexico takes the right upper limb as a sample.
1. Comparative example one
Reagent C was replaced with the following: collagenase I was present at a concentration of 2g/100ml with the other co-reagent C.
The procedure is as in steps 1, 2 and 3 of example 2.
Skin tissue has a cell death rate of about 60% and contains more cell debris.
2. Comparative example two
Reagent D was replaced with the following reagent: collagenase I was present at a concentration of 5g/100ml, with the other reagents D.
The procedure is as in steps 1, 2 and 4 of example 2.
Muscle tissue has serious cell deformation, most cells have no normal cell morphology, and more fragments exist in digestive juice, and the cell death rate is about 40%.
3. Comparative example three
Reagent F was replaced with the following: collagenase II was present at a concentration of 2g/100ml, with the other co-reagent F.
The procedure is as in steps 1, 2 and 5 of example 2.
Skeletal tissue, poor cell status, and cell death rate of about 37%.
Claims (1)
1. A method for dissociating an amphibian tissue to obtain single cells, comprising the steps of:
(1) Placing the sample tissue in a reagent B, soaking for 10-15min at room temperature, separating skin tissue, shearing, and treating with a reagent C for 1-3h;
(2) After the step (1) is completed, taking muscle tissues from the residual tissues, shearing, adding a reagent D for treatment for 1-2h, and then adding a reagent E for treatment for 20min;
(3) After the step (1) is completed, bone tissue is taken from the rest tissue, washed by the reagent A, chopped and treated by the reagent F for 1.5-2 hours;
the reagent B is a metal ion chelating agent solution, and contains 0.05-0.15g/100ml of metal ion chelating agent;
the reagent C consists of collagenase I, dispase II and a reagent A; in the reagent C, the concentration of collagenase I is 43000-53000U/100ml, and the concentration of dispase II is 90-110units/100ml;
the reagent D consists of collagenase I and a reagent A; in the reagent D, the concentration of collagenase I is 55000-65000U/100ml;
the reagent E consists of trypsin, a metal ion chelating agent and a reagent A; in the reagent E, the concentration of trypsin is 120000-180000USP U/100ml, and the concentration of the metal ion chelating agent is 0.03-0.07g/100ml;
the reagent F consists of collagenase II and a reagent A; in the reagent F, the concentration of collagenase II is 50000-55000U/100ml;
the reagent A consists of gentamicin sulfate, penicillin, streptomycin, DPBS and enzyme-free water; the reagent A contains 3-4mg/100ml gentamicin sulfate, 80-120 units/ml penicillin and 80-120 mug/ml streptomycin;
the amphibian is an salamander in mexico.
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