CN117025510A - Composition for cracking mouse pancreatic tissue, and cracking method and application of mouse pancreatic tissue - Google Patents
Composition for cracking mouse pancreatic tissue, and cracking method and application of mouse pancreatic tissue Download PDFInfo
- Publication number
- CN117025510A CN117025510A CN202310949088.0A CN202310949088A CN117025510A CN 117025510 A CN117025510 A CN 117025510A CN 202310949088 A CN202310949088 A CN 202310949088A CN 117025510 A CN117025510 A CN 117025510A
- Authority
- CN
- China
- Prior art keywords
- lysis
- sample
- collagenase
- pancreatic tissue
- composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000004923 pancreatic tissue Anatomy 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 24
- 239000000203 mixture Substances 0.000 title claims abstract description 22
- 238000005336 cracking Methods 0.000 title claims description 11
- 230000009089 cytolysis Effects 0.000 claims abstract description 31
- 241000699666 Mus <mouse, genus> Species 0.000 claims abstract description 28
- 108090000631 Trypsin Proteins 0.000 claims abstract description 24
- 102000004142 Trypsin Human genes 0.000 claims abstract description 24
- 239000012588 trypsin Substances 0.000 claims abstract description 24
- 102000029816 Collagenase Human genes 0.000 claims abstract description 22
- 108060005980 Collagenase Proteins 0.000 claims abstract description 22
- 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 22
- 229960002424 collagenase Drugs 0.000 claims abstract description 22
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 claims abstract description 21
- 241000699670 Mus sp. Species 0.000 claims abstract description 19
- 210000001519 tissue Anatomy 0.000 claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims description 29
- 108090000790 Enzymes Proteins 0.000 claims description 29
- 229940088598 enzyme Drugs 0.000 claims description 29
- 210000004027 cell Anatomy 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 26
- 239000006228 supernatant Substances 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 15
- 239000012091 fetal bovine serum Substances 0.000 claims description 12
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 238000003776 cleavage reaction Methods 0.000 claims description 11
- 230000007017 scission Effects 0.000 claims description 11
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 10
- 239000012634 fragment Substances 0.000 claims description 9
- 230000029087 digestion Effects 0.000 claims description 8
- 239000012139 lysis buffer Substances 0.000 claims description 8
- 238000001556 precipitation Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 6
- 239000013049 sediment Substances 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 210000003743 erythrocyte Anatomy 0.000 claims description 5
- 239000006166 lysate Substances 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 238000012163 sequencing technique Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 230000002934 lysing effect Effects 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 6
- 230000000052 comparative effect Effects 0.000 description 13
- 238000001514 detection method Methods 0.000 description 6
- 210000000496 pancreas Anatomy 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 4
- 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 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 101000577181 Aspergillus oryzae (strain ATCC 42149 / RIB 40) Extracellular metalloproteinase NpI Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000007418 data mining Methods 0.000 description 1
- 230000032798 delamination Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 108010007093 dispase Proteins 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000007481 next generation sequencing Methods 0.000 description 1
- 210000001819 pancreatic juice Anatomy 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012174 single-cell RNA sequencing Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000011222 transcriptome analysis Methods 0.000 description 1
- 231100000747 viability assay Toxicity 0.000 description 1
- 238000003026 viability measurement method Methods 0.000 description 1
Classifications
-
- 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
- C12N5/0676—Pancreatic cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a composition for mouse pancreatic tissue lysis, a method for preparing single cells by tissue lysis and application thereof. When collagenase I, collagenase IV, neutral proteinase I and trypsin are matched with a specific working concentration to prepare single cells by splitting the pancreatic tissues of the mice, the splitting time of the pancreatic tissues of the mice can be shortened as a whole, the full splitting is promoted, the cell activity rate after splitting is effectively improved, and the requirement of single cells on a machine is fully met.
Description
Technical Field
The invention relates to the technical field of single cell detection, in particular to a composition for mouse pancreatic tissue lysis, a mouse pancreatic tissue lysis method and application.
Background
Pancreas is one of the important organs in human, is the second large gland of human body, has complex structure, numerous functions, important endocrine and exocrine functions and complex regulation mechanism. Pancreatic juice secreted by pancreas plays an indispensable role in digestion and absorption of food, pancreas can secrete insulin, which is an important substance in human sugar metabolism, and diabetes may be caused if insulin secretion is insufficient.
The next generation sequencing technology is a powerful, cost-effective tool for performing high throughput transcriptome analysis, and by analyzing transcriptomes with surprising and unprecedented depth and accuracy, thousands of new transcript variants and isoforms have been demonstrated to be expressed in mammalian tissues or organs, which greatly accelerates our understanding of mammalian cell gene expression, regulation, and network complexity. Single-cell RNA sequencing is a novel high-throughput sequencing technology, can acquire a transcription map of each cell, consists of single-cell capturing, CDNA library preparation, RNA sequencing and data mining, and pancreatic single-cell sequencing can help the medical community to know the physiological and pathological processes of pancreas more deeply, and provides important basic research support for treating and preventing related diseases.
Among them, the most critical step in single cell sequencing is tissue dissociation, which directly affects data quality, resulting in poor results, and thus it is important to find an optimal dissociation scheme for a specific sample.
At present, collagen is conventionally adopted for enzymolysis of pancreas, and the problems of long enzymolysis time, low enzymolysis efficiency, low cell activity rate and the like are unfavorable for subsequent research, so that an efficient and stable method is urgently needed for tissue dissociation.
Disclosure of Invention
Based on the above, it is necessary to provide a composition for cleavage of mouse pancreatic tissue, a method for cleavage of mouse pancreatic tissue and application thereof, so as to improve the enzymolysis efficiency and increase the cell viability.
The invention adopts the following technical scheme:
in a first aspect, the invention provides a composition for use in the cleavage of pancreatic tissue in a mouse comprising collagenase I, collagenase IV, neutral proteinase I and trypsin.
Specifically, the working concentration ratio of collagenase I, collagenase IV, neutral proteinase I and trypsin is (0.1-10) mg/mL (0.1-1) mg/mL.
Preferably, the working concentration ratio of collagenase I, collagenase IV, neutral proteinase I and trypsin is (0.1-5) mg/mL, (0.1-5) mg/mL and (0.1-0.5) mg/mL.
More preferably, the working concentration ratio of collagenase I, collagenase IV, neutral proteinase I and trypsin is 0.5mg/mL, 2mg/mL, 0.8mg/mL, 0.25mg/mL.
Further, the composition for the lysis of mouse pancreatic tissue further comprises a lysis buffer. Preferably, the lysis buffer is one of RPMI1640 medium buffer containing 5% fetal bovine serum, phosphate buffer containing 5% fetal bovine serum, and erythrocyte lysate.
In a second aspect, the invention may also provide a lysis reagent for the lysis of mouse pancreatic tissue, comprising a composition for the lysis of mouse pancreatic tissue as described in the first aspect.
In a third aspect, the present invention also provides a method for lysing pancreatic tissue of a mouse, comprising the steps of:
dispersing collagenase I, collagenase IV and neutral proteinase I in a lysis buffer solution to obtain a mixed enzyme solution 1; dispersing trypsin in a lysis buffer to obtain a mixed enzyme solution 2;
cutting the pancreatic tissue of the mouse into pieces, placing the pieces in the mixed enzyme solution 1, performing digestion reaction for 20min at 37 ℃, standing and layering to obtain a supernatant 1 sample and a lower tissue fragment 1 sample;
mixing the tissue fragment 1 sample with the mixed enzyme solution 2, performing digestion reaction at 4 ℃ for 5-10min, standing and layering to obtain a supernatant 2 sample;
mixing the clear liquid 1 sample and the clear liquid 2 sample, filtering by using a 40 mu M cell sieve, centrifuging the filtrate at 4 ℃ and 500g, and removing the supernatant to obtain a first-order sediment sample;
adding phosphate buffer solution into the primary precipitation sample for resuspension, adding erythrocyte lysate for cracking, and centrifuging to remove supernatant to obtain a secondary precipitation sample; and adding phosphate buffer solution into the secondary sediment sample for resuspension, centrifuging to remove supernatant, and resuspension sediment to obtain the mouse pancreatic single-cell sample.
Further, before mixing the clear liquid 1 sample and the clear liquid 2 sample, further comprising:
transferring the clear liquid 1 sample into a centrifuge tube, and adding an equal volume of RPMI1640 containing 5% fetal bovine serum to terminate the reaction;
the supernatant 2 samples were transferred to centrifuge tubes and the reaction was stopped by adding an equal volume of RPMI1640 containing 5% fetal bovine serum.
The invention also provides the composition for the pancreatic tissue lysis of mice or the application of the tissue lysis reagent in a single cell sequencing method.
Compared with the prior art, the invention has the core advantages that:
according to the invention, a large number of experiments are carried out for screening and exploration, and the fact that collagenase I, collagenase IV, neutral proteinase I and trypsin are adopted to be matched with each other in a specific working concentration for preparing single cells through pancreatic tissue lysis of mice is found, so that the time for the pancreatic tissue lysis of the mice can be shortened as a whole, the tissue lysis is promoted to be sufficient, the cell activity rate after the lysis is effectively improved, and the requirement of single cells on machine is fully met.
Drawings
FIG. 1 is a graph showing the examination of pancreatic single-cell samples prepared in example 1 and comparative examples 1 to 6.
Detailed Description
The technical conception of the invention is to improve the method for cracking the pancreatic tissue of the mouse into single cells so as to shorten the cracking time of the pancreatic tissue of the mouse, promote the full cracking and improve the cell viability after the cracking.
The composition for the cleavage of the pancreatic tissue of the mice screened by the invention can be prepared into mixed enzymolysis liquid, preferably Collagenase I (C1 enzyme for short), collagenase IV (C4 enzyme for short), neutral proteinase I (dispase I for short, d1 enzyme), trypsin EDTA (T enzyme for short) and buffer liquid.
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art. The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
Example 1
The embodiment provides a method for preparing single cells by lysing pancreatic tissues of mice, which comprises the following steps:
s1, preparing a cracking reagent:
first, an RPMI1640 medium buffer containing 5% FBS was prepared.
Collagenase I, collagenase IV and neutral proteinase I are uniformly dispersed in 10mL of RPMI1640 culture medium buffer solution containing 5% FBS, and are fully and uniformly mixed to obtain a mixed enzyme solution 1 containing 0.5mg/mL collagenase I, 2mg/mL collagenase IV and 0.8mg/mL neutral proteinase I.
Trypsin was dispersed in RPMI1640 medium buffer containing 5% fbs to give mixed enzyme solution 2 containing 0.25mg/mL trypsin.
S2, tissue digestion and lysis:
the pancreas tissue of the mice is uniformly diced, the dicing size is about 1mm, the pancreas tissue is placed in 10mL of mixed enzyme solution 1, digested and reacted for 20min at 37 ℃, and the pancreas tissue is stood for layering, so that a supernatant fluid 1 sample and a lower tissue fragment 1 sample are obtained.
The supernatant 1 sample was transferred to a new centrifuge tube and the reaction was stopped by adding an equal volume of RPMI1640 containing 5% fetal bovine serum.
Mixing the lower layer tissue fragment 1 sample with the mixed enzyme solution 2, performing digestion reaction for 10min at 4 ℃, standing for delamination to obtain a supernatant liquid 2 sample and a lower layer tissue fragment 2 sample, wherein the tissue fragments in the lower layer tissue fragment 2 sample are basically digested.
The supernatant 2 samples were transferred to a new centrifuge tube and the reaction was stopped by adding an equal volume of RPMI1640 medium buffer containing 5% fetal bovine serum.
S3, preparing a single cell sample by re-suspension:
combining the clear liquid 1 sample and the clear liquid 2 sample, filtering by a 40 mu M cell sieve, centrifuging the filtrate at 4 ℃ and 500g for 10min, and removing the supernatant to obtain a first-order precipitation sample.
Adding 1mL of 1XPBS buffer solution (phosphate buffer solution) into the primary precipitation sample for resuspension, adding an equal volume of 1X erythrocyte lysate for further lysis after resuspension, carrying out lysis on ice for 3min, centrifuging at 4 ℃ under 500g for 5min, and removing the supernatant to obtain a secondary precipitation sample. And adding 1mL of 1XPBS buffer solution to the secondary precipitation sample for resuspension, centrifuging again, removing supernatant, and adding 1mL of 1XPBS buffer solution for resuspension to obtain the single cell sample.
Finally, the single cell sample obtained was subjected to a counting analysis using a cytometer.
Comparative example 1
This comparative example provides a method for the cleavage of pancreatic tissue in mice, the method steps of which are substantially the same as those of example 1, except that in steps S1 and S2:
no mixed enzyme solution 1 was prepared, and the pancreatic tissue of the mice was diced and digested at 37℃for 30min using only 0.25mg/mL trypsin (Trypsin EDTA) as an enzymatic hydrolysate.
Comparative example 2
This comparative example provides a method for pancreatic tissue lysis in mice, the method steps of which are substantially the same as in example 1, except that in steps S1 and S2:
no mixed enzyme solution 1 was prepared, and only 0.25mg/ml Trypsin EDTA was used as an enzymatic hydrolysate, and the pancreatic tissue of the mice was diced and digested at 4℃for 16 hours.
Comparative example 3
This comparative example provides a method for the cleavage of pancreatic tissue in mice, the method steps of which are substantially the same as those of example 1, except that in steps S1 and S2:
no mixed enzyme solution 2 was prepared, and the mouse pancreatic tissue was diced and digested at 37℃for 30min using only mixed enzyme solution 1 containing 0.5mg/mL collagenase I, 2mg/mL collagenase IV and 0.8mg/mL neutral proteinase I.
Comparative example 4
This comparative example provides a method for the cleavage of pancreatic tissue in mice, the method steps of which are substantially the same as those of example 1, except that in step S1:
the mixed enzyme solution 1 contains 0.5mg/mL collagenase I and 2mg/mL collagenase IV (without neutral proteinase I);
the enzyme mixture 2 contained 0.25mg/mL trypsin.
Comparative example 5
This comparative example provides a method for the cleavage of pancreatic tissue in mice, the method steps of which are substantially the same as those of example 1, except that in step S1:
the mixed enzyme solution 1 contains 0.5mg/mL collagenase I and 0.8mg/mL neutral proteinase I (without collagenase IV);
the enzyme mixture 2 contained 0.25mg/mL trypsin.
Comparative example 6
This example provides a method for the cleavage of pancreatic tissue in mice, which comprises substantially the same steps as in example 1, except that in step S1:
the mixed enzyme solution 1 contains 0.5mg/mL collagenase I and 2mg/mL collagenase IV (without neutral proteinase I);
the enzyme mixture 2 contained 0.25mg/mL trypsin.
Cell count detection and cell viability detection were performed on single cell samples prepared in example 1 and comparative examples 1 to 6, respectively.
The cell count detection method of the single cell sample comprises the following steps: mu.L of the single cell suspension was pipetted and mixed well with 10ul AO/Pi dye solution and counted with a cytometer.
The method for detecting the cell viability of the single cell sample comprises the following steps: mu.L of the single cell suspension was pipetted and mixed well with 10ul AO/Pi dye solution and then subjected to viability assay using a cytometer.
The statistics of the detection results are shown in fig. 1 and the following table:
single cell sample detection result statistical table
Further worth noting is that the inventors team, through extensive experimental investigation, also found that:
1) Trypsin alone has a certain lysis effect, but the treatment time is long and the number of cells obtained is small.
2) When mixed enzyme solution 1 (collagenase I, collagenase IV and neutral protease I) was used alone, the tissue mass was not significantly reduced, the number of cells obtained was very small, only 95 cells per microliter, and the activity was relatively low, only 80%.
3) When the mixed enzyme liquid 1 adopts any two of collagenase I, collagenase IV and neutral proteinase I, the mixed enzyme liquid 2 adopts trypsin, and the cell activity rate prepared by tissue digestion and cleavage is lower than 90%.
4) When the composition for splitting the pancreatic tissue of the mouse is applied, the working concentration of collagenase I is controlled to be (0.1-10) mg/mL, the working concentration of collagenase IV is controlled to be (0.1-10) mg/mL, the working concentration of neutral proteinase I is controlled to be (0.1-10) mg/mL, and the working concentration of trypsin is controlled to be (0.1-1) mg/mL.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A composition for use in the lysis of mouse pancreatic tissue comprising collagenase i, collagenase iv, neutral proteinase i and trypsin.
2. The composition for use in pancreatic tissue lysis in mice according to claim 1 wherein the working concentration ratio of collagenase i, collagenase iv, neutral proteinase i and trypsin is (0.1-10) mg/mL, (0.1-1) mg/mL.
3. The composition for use in the cleavage of pancreatic tissue in mice according to claim 2, wherein the working concentration ratio of collagenase i, collagenase iv, neutral proteinase i and trypsin is (0.1-5) mg/mL, (0.1-0.5) mg/mL.
4. A composition for use in the lysis of mouse pancreatic tissue according to claim 3 wherein the working concentration ratio of collagenase i, collagenase iv, neutral proteinase i and trypsin is 0.5mg/mL to 2mg/mL to 0.8mg/mL to 0.25mg/mL.
5. The composition for use in the lysis of mouse pancreatic tissue of any of claims 1 to 4, further comprising a lysis buffer.
6. The composition for use in the lysis of pancreatic tissue of mice according to claim 5, wherein the lysis buffer is one of RPMI1640 medium buffer containing 5% fetal bovine serum, phosphate buffer containing 5% fetal bovine serum, and erythrocyte lysate.
7. A lysis reagent for the lysis of mouse pancreatic tissue, characterized by comprising the composition for the lysis of mouse pancreatic tissue according to any one of claims 1 to 6.
8. A method for lysing pancreatic tissue of a mouse, comprising the steps of:
dispersing collagenase I, collagenase IV and neutral proteinase I in a lysis buffer solution to obtain a mixed enzyme solution 1; dispersing trypsin in a lysis buffer to obtain a mixed enzyme solution 2;
cutting the pancreatic tissue of the mouse into pieces, placing the pieces in the mixed enzyme solution 1, performing digestion reaction for 20min at 37 ℃, standing and layering to obtain a supernatant 1 sample and a lower tissue fragment 1 sample;
mixing the tissue fragment 1 sample with the mixed enzyme solution 2, performing digestion reaction at 4 ℃ for 5-10min, standing and layering to obtain a supernatant 2 sample;
mixing the clear liquid 1 sample and the clear liquid 2 sample, filtering by using a 40 mu M cell sieve, centrifuging the filtrate at 4 ℃ and 500g, and removing the supernatant to obtain a first-order sediment sample;
adding phosphate buffer solution into the primary precipitation sample for resuspension, adding erythrocyte lysate for cracking, and centrifuging to remove supernatant to obtain a secondary precipitation sample; and adding phosphate buffer solution into the secondary sediment sample for resuspension, centrifuging to remove supernatant, and resuspension sediment to obtain the mouse pancreatic single-cell sample.
9. The method of preparing single cells by tissue lysis according to claim 8, further comprising, prior to mixing the clear 1 sample and the clear 2 sample:
transferring the clear liquid 1 sample into a centrifuge tube, and adding an equal volume of RPMI1640 containing 5% fetal bovine serum to terminate the reaction;
the supernatant 2 samples were transferred to centrifuge tubes and the reaction was stopped by adding an equal volume of RPMI1640 containing 5% fetal bovine serum.
10. Use of a composition for the lysis of mouse pancreatic tissue according to any of claims 1 to 6 or a lysis reagent for the lysis of mouse pancreatic tissue according to claim 7 in a single cell sequencing method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310949088.0A CN117025510A (en) | 2023-07-30 | 2023-07-30 | Composition for cracking mouse pancreatic tissue, and cracking method and application of mouse pancreatic tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310949088.0A CN117025510A (en) | 2023-07-30 | 2023-07-30 | Composition for cracking mouse pancreatic tissue, and cracking method and application of mouse pancreatic tissue |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117025510A true CN117025510A (en) | 2023-11-10 |
Family
ID=88629091
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310949088.0A Pending CN117025510A (en) | 2023-07-30 | 2023-07-30 | Composition for cracking mouse pancreatic tissue, and cracking method and application of mouse pancreatic tissue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117025510A (en) |
-
2023
- 2023-07-30 CN CN202310949088.0A patent/CN117025510A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111088222B (en) | Preparation method of single-cell suspension of adipose tissue | |
| CN106701995A (en) | Method for cell quality control through unicellular transcriptome sequencing | |
| CN115074310B (en) | Method for simultaneously preparing single-cell suspension and single-cell nuclear suspension based on same lung tissue sample and application | |
| CN115369071B (en) | Universal method for dissociation of different tissues | |
| CN113604539B (en) | Low-temperature dissociation kit suitable for single-cell sequencing and application thereof | |
| CN113186156A (en) | Method for efficiently obtaining single cells in adipose tissue | |
| CN114045255B (en) | Method for separating and culturing tubular epithelial cells | |
| CN117025510A (en) | Composition for cracking mouse pancreatic tissue, and cracking method and application of mouse pancreatic tissue | |
| CN116396921B (en) | Kit special for pancreatic tissue digestion and pancreatic tissue digestion method | |
| CN110468180A (en) | Plasma dna library and its construction method | |
| CN116948955A (en) | Preparation method of human periosteum single cell suspension | |
| CN115322956A (en) | Kit for dissociation of different tissues and dissociation method thereof | |
| CN116376807A (en) | Composition for tissue lysis and method for preparing single cells by tissue lysis and application | |
| CN111349603B (en) | Dissociation method of breast cancer clinical puncture sample | |
| CN111518745A (en) | Preparation method and detection method of human parathyroid single cell suspension | |
| CN114277093A (en) | Method for extracting plant cell nucleus | |
| CN114591883A (en) | Preparation method of single cell suspension of aortic cells | |
| CN115725690A (en) | Solution for separating adipose cell nucleoplasm and application thereof | |
| CN113265371B (en) | Preparation method of efficient human kidney single cell suspension | |
| CN117586956A (en) | Dissociation solution combination, kit and method for preparing single cell suspension of human breast cancer tissue | |
| CN111575229B (en) | Separation method of placenta decidua stem cells | |
| CN111607649A (en) | Lung cancer fusion gene nucleic acid detection quality control product based on CRSIPR-Cas9 technology and preparation method thereof | |
| CN114990056B (en) | Separation and purification method of high-purity pig fat progenitor cells | |
| CN118345026A (en) | Preparation and application of universal high-activity mouse pancreas in-vitro tissue single cell suspension | |
| CN117757730B (en) | Kit special for digestion of human heart tissue, digestion method and detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |