CN107641615A - The immunocyte separation method of infiltration in a kind of mice pancreatic - Google Patents
The immunocyte separation method of infiltration in a kind of mice pancreatic Download PDFInfo
- Publication number
- CN107641615A CN107641615A CN201710870122.XA CN201710870122A CN107641615A CN 107641615 A CN107641615 A CN 107641615A CN 201710870122 A CN201710870122 A CN 201710870122A CN 107641615 A CN107641615 A CN 107641615A
- Authority
- CN
- China
- Prior art keywords
- cell
- pancreatic
- positive
- percoll
- infiltration
- 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
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The immunocyte separation side of infiltration in a kind of mice pancreatic.Method：With clostridiopetidase A IV, after DNA enzymatic I and RPMI1640 mixture slaking liquid pancreas digested tissue, filtered by 100 μm of nylon leaching nets, by density gradient centrifugation, collect intermediate layer cell after centrifugation, analysis cell pick-up rate, survival rate and cell category.As a result：Every mouse can obtain (3.0 ± 1.2) x105Individual cell；Isolated Cell viability is (96.7 ± 2.2) %；Through flow cytometry, CD45 positive T cells content is (97.1 ± 3.5) %, CD3 positive T cells content is (61.2 ± 5.7) %, CD4 positive T cells content is (49.1 ± 5.1) %, CD8 positive T cells content is that (47.2 ± 5.3) %, CD19 positive B-cells content is (32.8 ± 4.3) %.Conclusion：This method is simplified, efficiently, stably, and the cell quantity obtained is more, motility rate is high, purity is high, can provide reliable method for the immunocyte correlative study of Isolation of pancreatic infiltration.
Description
Technical field
The application is related to a kind of tissue inner cell separation method, the immunocyte point infiltrated in particularly a kind of mice pancreatic
From method.
Background technology
Pancreas is all closely bound up with life as one of most important organ in human body, its physiological action and pathological change.
In recent years, it is more and more so as to also occur with the improvement of people's life quality, the dietary structure of people also occurs to change
Pancreatic disease.The incidence of disease of pancreatic disease has the trend of rising in China or even world wide at present.Relevant pancreas
The report of disease, acute and chronic pancreatitis, idiopathic pancreatitis, autoimmune pancreatitis, cancer of pancreas etc. is more and more, pancreas
Gland disease is gradually caused attention.Many researchs of the researcher to pancreas focus mostly on to cancer of pancreas Signal Transduction path
Research, to cancer of pancreas fall ill Study on Molecular Mechanism, to molecule genetics research of cancer of pancreas etc..Therefore infiltration in Isolation of pancreatic
The important foundation that immunocyte turns into a variety of researchs works.But currently without the separation side that immunocyte is infiltrated in the pancreas of system
Method, then time-consuming, yield is low, step is complicated for the separation method of other existing histoorgan inner cells, and is not used to pancreas
The separation of inner cell.
The content of the invention
For existing separation method, time-consuming, yield is low, step is complicated and is not used to infiltrate immunocyte in pancreas
Defect, the present invention provide a set of simplification, the immunocyte separation method of the interior infiltration of mice pancreatic efficiently, stable.Specifically, this
The immunocyte separation method of infiltration, comprises the following steps in the mice pancreatic of invention：
(1) preparation of pancreatic tissue block：Mouse is put to death, takes out pancreas, 4 DEG C is placed in and phosphate buffer solution is housed
3.5cm diameters are positioned over culture dish on ice, are cleaned 1-2 times with phosphate buffer solution, move in new 3.5cm culture dishes,
Shred as needed to 1mm2 uniform volume block；
(2) separation of pancreatic tissue block and digestion：To be put into the pancreatic tissue block shredded 0.5mg/ml clostridiopetidase As IV and
In 0.01mg/ml DNA enzymatic I digestive juices, it is placed in 37 DEG C of constant-temperature tables after digesting 1.5h, takes out, suction out digestive juice, crosses 100 μm
Nylon leaching net into new 15ml centrifuge tubes, 1500rpm/5min, collect cell；
(3) density gradient centrifugation：4ml80% percoll solution is added in 15ml centrifuge tube, centrifuge tube tilts
60 degree, then the cell being resuspended with 8ml40% percoll solution is gently slowly added along tube wall and flow to 80%
On the liquid level of percoll solution, the percoll solution of the 4ml80% and 8ml40% are by RPMI-1640 complete mediums
Dilute, visible clearly interface between two layers of liquid；25 degree, 400g, 25min, raising speed 1, reduction of speed be 0 under conditions of from
The heart；Visible interface has the cell of one layer of fuzzy shape after centrifugation；Draw a little liquid of the superiors to discard, inhale interface intermediate cell
Layer liquid adds phosphate buffer solution to 15ml, mixing of turning upside down into new 15ml centrifuge tubes；Centrifugation, normal temperature,
1500rpm/5min, abandoning supernatant, it is resuspended and is precipitated with phosphate buffer solution, produce required cell.
Mice pancreatic lymphocyte is extracted using enzymic digestion, extraction step is simplified, efficiently, stably, obtains the quantity of cell
More, motility rate height, purity are high, and the molecular immunology mechanism for research pancreas related inflammation or tumor disease provides reliable method, is
Solid foundation is established in illustrating for pancreatic disease pathogenesis.
Brief description of the drawings
Fig. 1 is the living cells quantity that trypan blue staining detection obtains the immunocyte of infiltration in mice pancreatic
Fig. 2 is the Cell viability that the detection of Annexin V/PI apoptosis reagent obtains the immunocyte of infiltration in mice pancreatic
Fig. 3 is that the detection of CD45, CD3, CD4, CD8, CD19 antibody labeling obtains the immunocyte of infiltration in mice pancreatic
The ratio of cell type
Embodiment
1 material and method
1.1 experimental animal:C57BL/6 male mouses, 6~8 week old, Shanghai Si Laike animal centers are purchased from, are raised in SPF levels
In Animal House.
1.2 main agents and equipment：
Reagent：RPMI1640 nutrient solutions (Gibco companies of the U.S.), hyclone (ICP, New Zealand), 1XPBS (phosphorus
Phthalate buffer, 0.01mol/L), anti-mouse CD45 monoclonal antibodies, anti-CD3 monoclonal antibodies, anti-mouse
CD4 monoclonal antibodies, anti-mouse CD8 monoclonal antibodies, anti-mouse CD19 monoclonal antibodies (U.S. BD)；Collagen
Enzyme IV digestive juices (50mg/ml)；DNA enzymatic I (1mg/ml)；
Percoll separating liquids etc..
Equipment：15ml, 50ml centrifuge tube；Tissue Culture Dish；Operating theater instruments；Eppendorf centrifuges, Niko microscopes,
BD FACSCantoTM II flow cytometers etc.
1.3 mice pancreatic separation of lymphocytes methods
1.3.1 the preparation of pancreatic tissue block：Mouse is put to death, takes out pancreas, 4 DEG C is placed in and phosphate buffer solution is housed
(it is positioned on ice) in 3.5cm (diameter) culture dish, is cleaned 1-2 times with phosphate buffer solution, moves to new 3.5cm culture dishes
In, shred as needed to modest size (1mm2) uniform volume block；
1.3.2 the separation of pancreatic tissue block and digestion：To be put into the pancreatic tissue block shredded 0.5mg/ml clostridiopetidase As IV and
In 0.01mg/ml DNA enzymatic I digestive juices, it is placed in 37 DEG C of constant-temperature tables after digesting 1.5h, takes out；Digestive juice is suctioned out, crosses 100 μm
Nylon leaching net into new 15ml centrifuge tubes, 1500rpm/5min, collect cell；
1.3.3 density gradient centrifugation：4ml 80% percoll solution (dilution of 1640 complete mediums) is added to
In 15ml centrifuge tube, centrifuge tube tilts to horizontal (centrifuge tube tilts about 60 degree), then will be molten with 8ml 40% percoll
The cell that liquid (dilution of 1640 complete mediums) is resuspended gently slowly adds the liquid level for the percoll solution for flowing to 80% along tube wall
On, visible clearly interface between two layers of liquid；Centrifugation (room temperature, 25 degree, 400g, 25min, raising speed 1, reduction of speed 0)；From
Visible interface has the cell of one layer of fuzzy shape after the heart；Draw a little liquid of the superiors to discard, inhale intermediate layer cell layer liquid extremely
In new 15ml centrifuge tubes, PBS to 15ml, mixing of turning upside down are added；Centrifuge (normal temperature, 1500rpm/5min), supernatant discarding
Liquid, it is resuspended and is precipitated with PBS, it is stand-by.
1.4 mice pancreatic lymphocyte counts：
The cell for separating resuspension is observed into isolated pancreatic tissue separation of lymphocytes under inverted phase contrast microscope
Situation, manually carry out cell count.
1.5 mice pancreatic lymphocyte viability examinations：
Blue decoration method identification of cell vigor is expected using platform.The μ l of 0.4% trypan blue solution 90 are added to 10 μ l cell suspensions
In, fully mix, 3min is incubated at 20 DEG C, in 3min, distinguish living cell counting and dead cell with tally, it is non-staining
For living cells.
1.6 mice pancreatic lymphocyte survival rate tests：
Detected using Annexin V/PI apoptosis reagent.2 times, 1500rpm, 4 DEG C centrifugations of cell are washed with the PBS of precooling
5min.Collect the cell of 1-3 × 105.Add 100 μ 1 × Binding of l Buffer and cell is resuspended.Add 5 μ l Annexin V-
FITC and 5 μ l Pl Staining Solution, is gently mixed.Lucifuge, room temperature reaction 10min.Add 400 μ l 1 ×
Binding Buffer, mix, use flow cytomery.
1.7 mice pancreatic lymphocyte CDs 45, CD3, CD4, CD8, CD19 positive cell content detection：
Cell count, after take 100 μ l (1x106 cells) cell re-suspension liquids in clean EP pipes, centrifugation cell,
After cell is resuspended in 1xPBS, anti-mouse each 0.5ul of CD45, CD3, CD4, CD8, CD19 antibody of FITC marks is added, is gently mixed
Even, 4 DEG C of lucifuges are incubated 20min.Afterwards plus 1ml 1xPBS, 4 DEG C of centrifugations, 1500rpm, 5min, supernatant is abandoned, be eventually adding
Through flow cytomery after 200ul1xPBS resuspensions.Collect 10000 cells and be used for data analysis, using the same of FITC marks
Type antibody is as negative control.
1.8 statistical method：Data carry out Treatment Analysis using Graphpad Prism5 statistical softwares, are represented with x ± s.
Single sample calculates cell yield and survival rate with the t methods of inspection.
2 results
2.1 mice pancreatic lymphocytes are observed
2.2 mice pancreatic lymphocyte quantities, vigor and characteristic：Using above-mentioned separation method, the pancreas of 2 mouse is isolated
Gland, every mouse can obtain 1-3x105Individual lymphocyte.Platform expects that blue stained cells vigor is higher, and dead cell is few, sees Fig. 1, no
Coloring for living cells
The Cell viability of 2.3 mice pancreatic lymphocytes：By flow cytomery, mouse is obtained using the above method
Pancreas lymphocyte motility rate be 96.7%, the motility rate of negative control is 98.1%.Wherein left lower quadrant is living cells, prompts institute
Apoptosis and dead cell are less in separation cell, see Fig. 2.
2.4 mice pancreatic lymphocyte CDs 45, CD3, CD4, CD8 and CD19 positive cell content：The above method is separated
Mice pancreatic lymphocyte be marked with CD45, CD3, CD4, CD8, CD19 positive antibody, pass through flow cytomery
Separation purity, using above-mentioned separation method obtain mice pancreatic lymphocyte in, CD45 positive cells content be 97.1%,
CD3 positive T cells content is that 61.2%, CD4 positive T cells content is that 49.1%, CD8 positive T cells content is 47.2%,
CD19 positive B-cells content is 32.8%, sees Fig. 3.
3. discuss
Cancer of pancreas is aggressive highest malignant tumour in digestive system, based on duct adenocarcinoma.Although with other tumours
Compare, pancreas cancer morbidity is relatively low, but due to its extremely low survival rate (being less than 5%), cancer of pancreas is turned into cancer related mortality
The fourth-largest main cause [9] of factor；Therefore research pancreas relevant disease is significant.And it is thin to separate acquisition mice pancreatic lymph
Born of the same parents establish solid foundation for research pancreas relevant disease and illustrating for pancreatic disease pathogenesis.We are by separating LPL's
Method is inspired, with reference to the LPL of the offers such as Sanos, Zhu Siying separation method and improved, to separating mouse pancreas
Lymphocyte, and the pancreas lymphocyte effect that observation analysis obtains, it is thin to be successfully separated out efficient, stable mice pancreatic lymph
Born of the same parents, this method have the advantages that simplified, efficient, stabilization, have good promotional value.
During separation, it has been found that influenceing the factor that mice pancreatic lymphocyte obtains has：Temperature：37 DEG C are shaken
Digestion process tissue in bed, separative efficiency can be improved, and remaining step keeps at low ambient temperatures, being easy to improve cellular activities
Rate.As shown in figure 3, it is 96.7% to detect Cell viability by flow cytometer showed.Digestion time：It should strictly control and disappear in digestion process
Change the time, prevent from digesting over or under.Density gradient centrifugation：Must be accurate in the Percoll working solutions concentration of preparation；Adding
When entering upper strata low concentration cell suspension separating liquid to high concentration percoll separating liquids, action will be light and keeps interface clear；Otherwise
Directly affect centrifugal effect.It should be noted during experiment：1. digest：General digestion time is about 1.5h, visually observes bottom of bottle by big
When graininess switchs to little particle translucent white boundless and indistinct shape, you can take out digestive juice.2. Percoll density gradient centrifugations：It is general to use
Centrifugal force is 400g, time 20-25min.Because density difference is little between different layers Percoll, therefore centrifuge accelerates, drop
It is slow when fast, it is steady.Our cell overwhelming majority to be separated gently remove upper strata Percoll at two layers of interface
The cell of interface locations is collected after liquid；Sometimes most cells are located in Percoll layers, then need successively to collect.3. trypan blue
Decoration method：Dyeing time is unsuitable oversize, otherwise living cells also due to stain accumulation and colour, make monitoring result relatively low；④
Annexin V/PI apoptosis reagent detects：Because Apoptosis is a quick process, it is proposed that sample enters as early as possible after dyeing
Row analysis.Annexin V-FITC and PI are light sensitive species, and lucifuge operation is paid attention to during dying operation.5. fluidic cell
Instrument detects：Should ensure that concentration of the sample before upper machine testing is 1X106Cell/ml, cell concentration is too low to influence whether detection knot
Fruit accuracy.Fully washed after fluorescent antibody staining, pay attention to mixing and centrifugal speed, reduce overlapping cell and cell fragment.If
Control sample is put, using the unrelated control and the Background control of fluorescence antibody matched with antibody sources homotype., should during result of determination
Pay attention to subtracting background fluorescence.Pay attention to lucifuge after dyeing, ensure the stabilization of cellular immunofluorescence.
Mice pancreatic lymphocyte is extracted using enzymic digestion, extraction step is simplified, efficiently, stably, obtains the quantity of cell
More, motility rate height, purity are high, provide reliable method for the immune research of research pancreas, are established for illustrating for pancreatic disease pathogenesis
Determine solid foundation.
Claims (1)
1. the immunocyte separation method of infiltration, comprises the following steps in a kind of mice pancreatic：
(1) preparation of pancreatic tissue block：Mouse is put to death, takes out pancreas, it is straight to be placed in 4 DEG C of 3.5cm equipped with phosphate buffer solution
Footpath is positioned over culture dish on ice, is cleaned 1-2 times with phosphate buffer solution, moves in new 3.5cm culture dishes, as needed
Shred to 1mm2 uniform volume block；
(2) separation of pancreatic tissue block and digestion：0.5mg/ml clostridiopetidase As IV and 0.01mg/ will be put into the pancreatic tissue block shredded
In ml DNA enzymatic I digestive juices, it is placed in 37 DEG C of constant-temperature tables after digesting 1.5h, takes out, suction out digestive juice, crosses 100 μm of nylon
Filter screen is into new 15ml centrifuge tubes, 1500rpm/5min, collects cell；
(3) density gradient centrifugation：4ml 80% percoll solution is added in 15ml centrifuge tube, centrifuge tube tilts 60
Degree, then the percoll for flowing to 80% gently will be slowly added along tube wall with the cell of 8ml 40% percoll solution resuspension
On the liquid level of solution, the percoll solution of the 8ml 40% is diluted by RPMI-1640 complete mediums, between two layers of liquid
It can be seen that clearly interface；25 degree, 400g, 25min, raising speed 1, reduction of speed be 0 under conditions of centrifuge；Visible boundary after centrifugation
There is the cell of one layer of fuzzy shape in face；Draw a little liquid of the superiors to discard, inhale interface intermediate cell layer liquid to new 15ml
In centrifuge tube, phosphate buffer solution is added to 15ml, mixing of turning upside down；Centrifugation, normal temperature, 1500rpm/5min, is discarded
Clear liquid, it is resuspended and is precipitated with phosphate buffer solution, produce required cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710870122.XA CN107641615A (en) | 2017-09-22 | 2017-09-22 | The immunocyte separation method of infiltration in a kind of mice pancreatic |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710870122.XA CN107641615A (en) | 2017-09-22 | 2017-09-22 | The immunocyte separation method of infiltration in a kind of mice pancreatic |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107641615A true CN107641615A (en) | 2018-01-30 |
Family
ID=61113540
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710870122.XA Pending CN107641615A (en) | 2017-09-22 | 2017-09-22 | The immunocyte separation method of infiltration in a kind of mice pancreatic |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107641615A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257330A (en) * | 2019-06-24 | 2019-09-20 | 浙江大学 | A kind of method of effective acquisition mouse lung panimmunity cell |
| CN112342193A (en) * | 2019-08-07 | 2021-02-09 | 四川大学华西医院 | Method for quantitatively separating and detecting intestinal muscle layer infiltrating immune cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705206A (en) * | 2009-11-25 | 2010-05-12 | 东北林业大学 | Novel highly efficient method for preparing mice pancreatic single cell suspension |
| CN104780939A (en) * | 2012-08-20 | 2015-07-15 | 弗雷德哈钦森癌症研究中心 | Method and compositions for cellular immunotherapy |
-
2017
- 2017-09-22 CN CN201710870122.XA patent/CN107641615A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705206A (en) * | 2009-11-25 | 2010-05-12 | 东北林业大学 | Novel highly efficient method for preparing mice pancreatic single cell suspension |
| CN104780939A (en) * | 2012-08-20 | 2015-07-15 | 弗雷德哈钦森癌症研究中心 | Method and compositions for cellular immunotherapy |
Non-Patent Citations (1)
| Title |
|---|
| 朱思莹等: "一种改良小鼠肠道黏膜固有层淋巴细胞分离方法的建立", 《武汉大学学报（医学版）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257330A (en) * | 2019-06-24 | 2019-09-20 | 浙江大学 | A kind of method of effective acquisition mouse lung panimmunity cell |
| CN112342193A (en) * | 2019-08-07 | 2021-02-09 | 四川大学华西医院 | Method for quantitatively separating and detecting intestinal muscle layer infiltrating immune cells |
| CN112342193B (en) * | 2019-08-07 | 2023-03-31 | 四川大学华西医院 | Method for quantitatively separating and detecting intestinal muscle layer infiltrating immune cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20190017919A1 (en) | Microfluidic Device And Method For Detecting Rare Cells | |
| KR101680619B1 (en) | Method for identification, selection and analysis of tumour cells | |
| US20110195413A1 (en) | Integrated Method for Enriching and Detecting Rare Cells from Biological Body Fluid Sample | |
| WO2017020617A1 (en) | Method and device for detecting circulating tumor cell | |
| CN107490672B (en) | method for rapidly analyzing crustacean hemolymph cell group and number and application | |
| CN107287107A (en) | A kind of circulating tumor cell separation equipment, system and method | |
| CN101533012A (en) | Method and apparatus for imaging target components in biological sample by using permanent magnet | |
| CN105087493B (en) | Three kinds of monoclonal antibody coupling immunomagnetic beads of combination are applied in tumour cell sorting | |
| CN106244553A (en) | The separation of circulating tumor cell and detection method | |
| CN109351370B (en) | Microfluidic chip and cell screening method | |
| CN109777775A (en) | A kind of circulating tumor cell separation method | |
| CN105987843A (en) | Method for isolating or detecting rare cell | |
| CN107641615A (en) | The immunocyte separation method of infiltration in a kind of mice pancreatic | |
| CN109187977A (en) | It is a kind of detect HER2 antigen different loci immunofluorescent reagent box and application | |
| Terstappen et al. | Flowcytometry‐Principles and Feasibility in Transfusion Medicine. Enumeration of Epithelial Derived Tumor Cells in Peripheral Blood | |
| CN108982839A (en) | Circulating tumor cell detection method based on immunomagnetic beads and flow cytometer | |
| CN110333358A (en) | A kind of method for building up of mice with acute lung injury lungs panimmunity cell characteristic map | |
| CN108865654A (en) | A kind of cell sorting device and method for separating | |
| CN101730738A (en) | Method according to viscoelasticity property separating bio object | |
| CN110628721A (en) | Isolated culture method and kit for circulating tumor cells | |
| CN113125738A (en) | Method for detecting circulating tumor cells | |
| CN108982874A (en) | It is a kind of detect human prostate cancer antigen PSA, AR-V7 immunofluorescent reagent box and application | |
| CN109073626A (en) | Acquisition methods are separated using the detection of the circulating tumor cell of cell proliferation method | |
| CN109187976A (en) | The immunofluorescence detection agent box of androgen receptor splicing variant AR-V7 and application | |
| CN114729055B (en) | Methods for detecting and isolating cell populations co-expressing CD45 and EpCAM and uses thereof |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180130 |
|
| RJ01 | Rejection of invention patent application after publication |