CN106676066A - Method of separating and purifying II-type innate lymphoid cells from animal lung tissue - Google Patents
Method of separating and purifying II-type innate lymphoid cells from animal lung tissue Download PDFInfo
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- CN106676066A CN106676066A CN201611263313.1A CN201611263313A CN106676066A CN 106676066 A CN106676066 A CN 106676066A CN 201611263313 A CN201611263313 A CN 201611263313A CN 106676066 A CN106676066 A CN 106676066A
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Abstract
The invention provides a method of separating and purifying II-type innate lymphoid cells from animal lung tissue. The method includes: taking the animal lung tissue, crushing, and adding I-type collagenase for digestion to obtain a lung tissue digestion solution; filtering the lung tissue digestion solution; separating to obtain lung tissue lymphoid cells through a Percoll method; using a II-type innate lymphoid cell specific antibody to mark the lung tissue lymphoid cells, and separating to obtain the II-type innate lymphoid cells through flow cytometry. By the method, high-activity II-type innate lymphoid cells can be separated and purified from mouse lung tissue.
Description
Technical field
The present invention is with regard to a kind of method that the intrinsic lymphocyte of II types is isolated and purified from animal lung tissue.
Background technology
In recent years, the further investigation with people to inherent immunity cell, it was found that the new cell subsets of a group.This kind of Asia
Group not antigen expressed specific recognition receptor (TCR or BCR), belongs to lymphocyte linage, with past people in development and form
NKT (NK) cell that finds and lymphoid tissue induction (LTi) cell be collectively known as intrinsic lymphocyte (ILC).Press
According to the sorting technique of complementary T (Th) cell, according to cytokine secretion and the difference of transcription factor expression, ILC families again may be used
It is divided into three major types：ILC1, ILC2 and ILC3.
The intrinsic lymphocyte of II type (Type II innate lymphoid cells, ILC2) is above-mentioned one of which
Newly discovered intrinsic lymphocyte population, is distributed mainly on the mucous membrane tissues such as lungs, intestinal, skin, in IL-25 and IL-33
Stimulation under can produce the Th2 cytokines such as IL-5 and IL-13.ILC2 is in respiratory tract anti-infectious immunity and anaphylaxis disease
Play a significant role in disease, thus receive much concern.There are some researches show, in respiratory anaphylactic disease morbidity early stage, ILC2 is
The important sources of Th2 cytokines, can regulate and control adaptive immune response；Simultaneously in parasiticide, virus infection and group
In knitting repair process, ILC2 also plays critical function.Therefore, the biological study of ILC2 is of great immediate significance.Although
Research in ILC2 association areas at present achieves great breakthrough, but because ILC2 cell quantities are few, cell surface lacks
The marker molecule of high special, thus still suffer from great technological challenge in terms of obtaining the cell and being used for research.Wherein, lung
Dirty is one of organ that ILC2 cells are found earliest, and lungs ILC2 mediates the II types of the disease early stages such as respiratory tract anaphylaxis reaction to exempt from
Epidemic disease response, it is the key technology that ILC2 biologys are studied by mouse model that ILC2 cells are precisely separating from mouse lung, so
And, the detailed description to mouse lung ILC2 cell isolation method is there is no at present.
The content of the invention
It is an object of the present invention to provide a kind of method that ILC2 cells are precisely separating in lungs from animal.
The invention provides a kind of method that the intrinsic lymphocyte of II types is isolated and purified from animal lung tissue, the method bag
Include：
Animal lung tissue is taken, is crushed, add type i collagen enzymic digestion, obtain lung tissue digestion solution；
Lung tissue digestion solution is filtered；
By the isolated lung tissue lymphocyte of Percoll methods；
With the lung tissue lymphocyte obtained by the intrinsic lymphocyte specific antibody labeling of II type, Jing cell streaming technologies
Sorting obtains the intrinsic lymphocyte of II type.
Specific embodiment of the invention, in the method for the present invention, the animal is mice.
Specific embodiment of the invention, is into 1~2mm by animal lungs tissue shear in the method for the present invention3Greatly
It is little to add type i collagen enzymic digestion.
Specific embodiment of the invention, in the method for the present invention, the lungs of every mice of correspondence, addition 3~
20ml concentration is the type i collagen enzymic digestion of 0.03~0.5% (m/v).
Specific embodiment of the invention, in the method for the present invention, adds the ring at 30~42 DEG C after type i collagen enzyme
Digest 10~90 minutes in border.
Specific embodiment of the invention, in the method for the present invention, lung tissue digestion solution is the screen cloth mistake of 200 mesh
Filter.
Specific embodiment of the invention, in the method for the present invention, the isolated lung tissue lymph of Percoll methods is thin
The concrete operations of born of the same parents are：
With the resuspended precipitations of 42%Percoll 3ml, then the cell suspension is added slowly to into 3ml 70%Percoll tables
Face, centrifugation 1260g 30 minutes.
Specific embodiment of the invention, in the method for the present invention, by four kinds of antibody from liver mononuclearcell
In carry out the flow cytometry sorting intrinsic lymphocyte of the type of purification II.Preferably, the antibody is FITC-anti mouse
KLRG1、PE-anti mouse Lineage Cocktail、PerCP.Cy5.5-anti mouse CD90.2、APC-anti
mouse IL33R.These antibody are commercially available, for example, be purchased from Biolegend companies.
In a specific embodiment of the present invention, the present invention's isolates and purifies the intrinsic lymph of II types from animal lung tissue
The method of cell includes：
Animal lung tissue is cut into into 1~2mm3Left and right fritter is put into piece of tissue in centrifuge tube plus tools of the 5ml without serum
The 0.1% collagenase I solution that the solution (such as incomplete DMEM or normal saline) for having physiological osmotic pressure is prepared, is placed in 37 DEG C of shakes
Swing 220rpm in case to digest 30 minutes；It is placed in and terminate on ice digestion, with 200 mesh sieve net filtrations, will stay on online lung tissue
Refilter after being lightly ground；
4 DEG C of 1060g are centrifuged 10 minutes, abandon supernatant；
Density gradient centrifugation：With the resuspended precipitations of 42%Percoll 3ml, then the cell suspension is added slowly to
3ml70%Percoll (GE Healthcare) surface, centrifugation 1260g 30 minutes；
Intermediate layer cell is taken to 15 milliliters of new centrifuge tubes, PBS is filled it up with, 1060g be centrifuged 10 minutes after abandoning supernatant；
Cell suspension adds rat blood serum block Fc receptors and mark fluorescent antibody：FITC-anti mouse KLRG1、
PE-anti mouse Lineage Cocktail、PerCP.Cy5.5-anti mouse CD90.2、APC-anti mouse
IL33R, 4 DEG C of lucifuges are incubated 15min；
PBS, 4 DEG C of 1060g × 10min are filled it up with, twice is washed；
With selected by flow cytometry apoptosis Lineage-IL33R+CD90.2+KLRG1+ cells, as ILC2 cells.
The method of the present invention, discharges lymphocyte and maintains lymphocyte to live to greatest extent with collagenase I from tissue
Property；By density gradient centrifugation, lymphocyte is opened with liver organization cell separation, it is thin so as to be enriched with the intrinsic lymph of lungs II types
Born of the same parents.The method can separate and be purified into height through facts have proved in a large number from normal or C57BL/6 mices with lungs inflammation
Purity, the intrinsic lymphocyte of highly active II types.
Description of the drawings
Fig. 1 represents Gate step of the intrinsic lymphocyte of II type in assorting room.
Fig. 2 shows the flow cytometry sorting experimental result of ILC2 cells.
Fig. 3 shows the function/activity identification result of the ILC2 cells obtained by sorting.
Fig. 4 A and Fig. 4 B show the ILC2 cell quantities that Jing this method can be obtained under different condition.
Specific embodiment
In order to be more clearly understood that the present invention, referring now to the following example and accompanying drawing the present invention is further described.Embodiment
It is only used for explaining and limiting the present invention never in any form.The experimental technique of unreceipted actual conditions is art in embodiment
Well known conventional method and normal condition, or according to the condition proposed by manufacturer.
Embodiment 1
Below experiment is both needed to sterile working.
(1) the normal or mouse lung tissue with lungs inflammation, is put in aseptic PBS and is placed on ice.
(2) tissue is cut into into 1~2mm3 or so fritters piece of tissue to be put in centrifuge tube plus the incomplete DM EM preparations of 5ml
Collagenase I solution (0.1%, i.e. 1mg/ml, sigma), screw lid be placed in 37 DEG C concussion casees in 220rpm digest 30 minutes
(visible tissue block disperses and loses the shape of block, that is, digest successfully).
After the completion of (3) the 3rd steps, it is placed in and terminate on ice digestion, with 200 mesh sieve net filtrations, will stay on online lung tissue
Refilter after being lightly ground.
(4) 4 DEG C of 1060g are centrifuged 10 minutes, abandon supernatant.(it is in addition to room temperature to be centrifuged below the step of density gradient centrifugation
It it is 4 DEG C).
(5) density gradient centrifugation：With the resuspended precipitations of 42%Percoll 3ml, 3ml 70%Percoll (GE are added slowly to
Healthcare) surface, centrifugation 1260g 30 minutes.
20ml 70%Percoll formula：12.6ml Percoll stock solutions (GE Healthcare)+
1.4ml 10×PBS+6ml 1×PBS
20ml 42%Percoll formula：7.56ml Percoll stock solutions (GE Healthcare)+
840ul 10×PBS+11.6ml 1×PBS
(6) take intermediate layer cell to 15 milliliters of new centrifuge tubes, PBS is filled it up with, 1060g be centrifuged 10 minutes after supernatant discarded
Liquid, then with 1 milliliter of PBS re-suspended cell.
(7) a small amount of cell is taken out with being counted after Trypan Blue.
(8) according to count results, the rat serum of cell suspension 10% suspension volume of addition for including 1,000,000 cells is taken
Clear block Fc receptors and mark fluorescent antibody：FITC-anti mouse KLRG1、PE-anti mouse Lineage
Cocktail, PerCP.Cy5.5-anti mouse CD90.2, (antibody is all from APC-anti mouse IL33R
Biolegend), 4 DEG C of lucifuges are incubated 15min.
(9) PBS, 4 DEG C of 1060g × 10min are filled it up with, twice is washed.
(10) selected by flow cytometry apoptosis Lineage is used-IL33R+CD90.2+KLRG1+Cell, as ILC2 cells.
Fig. 1 represents Gate step of the intrinsic lymphocyte of II type in assorting room：
Lung mononuclearcell → CD45+→KLRG1+CD90+→Lineage-IL-33R+
Fig. 2 shows experimental result, separates according to this method and can clearly detect depositing for ILC2 cells after lungs lymphocyte
The position that it is shown on flow cytometer is as shown in Figure 2.The Jing inspection policies carry out airflow classification, per only adult
Normal mouse can obtain the intrinsic lymphocyte of 4000-10000 II type, and mice can obtain bigger under lungs inflammatory conditions
The cell of quantity, particular number is depending on severity of inflammation.Cell obtained by sorting, in vitro with the training of 50000/mL density
When foster, with mIL-33 (such as 50ng/mL mIL-33) stimulate 2 days after can produce substantial amounts of Th2 cytokines such as IL-5 with
IL-13, but Th1 cytokines such as IFN-γ (Fig. 3) can not be produced, represent that the cell has preferably activity.
Embodiment 2
The lungs Jing method of the present invention of normal mouse, the collagenase I with several variable concentrations of normal saline is molten
Liquid digests, and as a result other operations show to separate and be purified into the intrinsic lymphocyte of highly active II types, institute's energy with embodiment 1
The intrinsic lymphocyte quantity of II types of acquisition is referring to Fig. 4 A.
Normal mouse divides after 80 micrograms Bo Laimei element collunarium induction lungs inflammation one week with reference to the method for embodiment 1
From the intrinsic lymphocyte of II types, as a result show to separate and be purified into the intrinsic lymphocyte of highly active II types, the II types of gained
Intrinsic lymphocyte quantity is referring to Fig. 4 B.
Claims (10)
1. a kind of method that the intrinsic lymphocyte of II types is isolated and purified from animal lung tissue, the method includes：
Animal lung tissue is taken, is crushed, add type i collagen enzymic digestion, obtain lung tissue digestion solution；
Lung tissue digestion solution is filtered；
By the isolated lung tissue lymphocyte of Percoll methods；
With the lung tissue lymphocyte obtained by the intrinsic lymphocyte specific antibody labeling of II type, the sorting of Jing cells streaming technology
Obtain the intrinsic lymphocyte of II type.
2. method according to claim 1, wherein, the animal is mice.
3. method according to claim 1, wherein, it is into 1~2mm by animal lungs tissue shear3Size adds I type glue
Protoenzyme digests.
4. method according to claim 1, wherein, the lungs of every mice of correspondence add 3~20ml concentration be 0.03~
The type i collagen enzymic digestion of 0.5% (m/v).
5. the method according to claim 1 or 4, wherein, add and digested in the environment after type i collagen enzyme between 30~42 DEG C
10~90 minutes.
6. method according to claim 1, wherein, lung tissue digestion solution is the screen filtration of 200 mesh.
7. method according to claim 1, wherein, the concrete operations of the isolated lung tissue lymphocyte of Percoll methods
For：
With the resuspended precipitations of 42%Percoll 3ml, then the cell suspension is added slowly to into 3ml 70%Percoll surfaces, is entered
Row centrifugation 1260g 30 minutes.
8. method according to claim 1, wherein, fluidic cell is carried out from liver mononuclearcell by four kinds of antibody
The intrinsic lymphocyte of the art sorting type of purification II.
9. the method according to claim 1 or 8, wherein, the antibody is FITC-anti mouse KLRG1, PE-anti
mouse Lineage Cocktail、PerCP.Cy5.5-anti mouse CD90.2、APC-anti mouse IL33R。
10. method according to claim 1, the method includes：
Animal lung tissue is cut into into 1~2mm3Left and right fritter by piece of tissue be put in centrifuge tube plus 5ml without serum with physiology
The 0.1% collagenase I solution that the solution (such as incomplete DMEM or normal saline) of osmotic pressure is prepared, is placed in 30~42 DEG C of concussions
110~220rpm digests 10~90 minutes in case；It is placed in and terminate on ice digestion, with 200 mesh sieve net filtrations, will stay on online
Lung tissue is refiltered after being lightly ground；
4 DEG C of 1060g are centrifuged 10 minutes, abandon supernatant；
Density gradient centrifugation：With the resuspended precipitations of 42%Percoll 3ml, then the cell suspension is added slowly to into 3ml70%
Percoll (GE Healthcare) surface, centrifugation 1260g 30 minutes；
Intermediate layer cell is taken to 15 milliliters of new centrifuge tubes, PBS is filled it up with, 1060g be centrifuged 10 minutes after abandoning supernatant；
Cell suspension adds rat blood serum block Fc receptors and mark fluorescent antibody：FITC-anti mouse KLRG1、PE-
anti mouse Lineage Cocktail、PerCP.Cy5.5-anti mouse CD90.2、APC-anti mouse
IL33R, 4 DEG C of lucifuges are incubated 15min；
PBS, 4 DEG C of 1060g × 10min are filled it up with, twice is washed；
With selected by flow cytometry apoptosis Lineage-IL33R+CD90.2+KLRG1+ cells, as ILC2 cells.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410820A (en) * | 2018-03-14 | 2018-08-17 | 南京鼓楼医院 | A method of isolating and purifying intrinsic lymphocyte from mouse intestinal mucosa |
| CN109010357A (en) * | 2018-02-12 | 2018-12-18 | 温州医科大学 | The construction method of acute lung injury inflammatory resolution animal model |
| CN110257330A (en) * | 2019-06-24 | 2019-09-20 | 浙江大学 | A kind of method of effective acquisition mouse lung panimmunity cell |
| CN110333358A (en) * | 2019-06-24 | 2019-10-15 | 浙江大学 | A kind of method for building up of mice with acute lung injury lungs panimmunity cell characteristic map |
| CN113061575A (en) * | 2021-03-25 | 2021-07-02 | 中国人民解放军陆军军医大学 | Method for separating and purifying inherent lymphoid cells from colon inherent layer of old mouse |
| CN113355273A (en) * | 2021-03-08 | 2021-09-07 | 安徽理工大学 | Method capable of quickly and effectively separating cells from cell suspension containing dust |
| CN115418350A (en) * | 2022-09-01 | 2022-12-02 | 中国科学技术大学 | Method for separating inherent lymphocytes from mouse brain membrane and application |
-
2016
- 2016-12-30 CN CN201611263313.1A patent/CN106676066A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| KAZUYO MORO等: "Isolation and analysis of group 2 innate lymphoid cells in mice", 《NATURE PROTOCOLS》 * |
| PHILIP A等: "Intrinsic functional defects of ILC2 impair innate allergic inflammation in PLZF-deficient mice", 《J ALLERGY CLIN IMMUNOL》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109010357A (en) * | 2018-02-12 | 2018-12-18 | 温州医科大学 | The construction method of acute lung injury inflammatory resolution animal model |
| CN108410820A (en) * | 2018-03-14 | 2018-08-17 | 南京鼓楼医院 | A method of isolating and purifying intrinsic lymphocyte from mouse intestinal mucosa |
| CN110257330A (en) * | 2019-06-24 | 2019-09-20 | 浙江大学 | A kind of method of effective acquisition mouse lung panimmunity cell |
| CN110333358A (en) * | 2019-06-24 | 2019-10-15 | 浙江大学 | A kind of method for building up of mice with acute lung injury lungs panimmunity cell characteristic map |
| CN113355273A (en) * | 2021-03-08 | 2021-09-07 | 安徽理工大学 | Method capable of quickly and effectively separating cells from cell suspension containing dust |
| CN113061575A (en) * | 2021-03-25 | 2021-07-02 | 中国人民解放军陆军军医大学 | Method for separating and purifying inherent lymphoid cells from colon inherent layer of old mouse |
| CN113061575B (en) * | 2021-03-25 | 2023-04-07 | 中国人民解放军陆军军医大学 | Method for separating and purifying inherent lymphoid cells from colon inherent layer of old mouse |
| CN115418350A (en) * | 2022-09-01 | 2022-12-02 | 中国科学技术大学 | Method for separating inherent lymphocytes from mouse brain membrane and application |
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Application publication date: 20170517 |