CN107748130A - A kind of preparation of animal hearts single cell suspension and detection method - Google Patents
A kind of preparation of animal hearts single cell suspension and detection method Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The invention discloses a kind of preparation of animal hearts single cell suspension and detection method, this method to include：Step 1：Make animal hearts ischemia-reperfusion injury model；Step 2：Animal isolated heart tissue is taken, washes off blood, heart digestive juice is added, shreds, mixes digestion；Step 3：The mixed solution that filtration step 2 obtains, centrifugation, supernatant is removed, terminate digestion, added phosphate buffer and suspend, added erythrocyte cracked liquid, centrifugation, remove supernatant, added phosphate buffer and suspend, obtain animal hearts single cell suspension.The preparation method of the present invention can reduce the destruction to cell, there is provided stablize, reliable single cell suspension preparation method, cell number is higher in the suspension of preparation, and the detection method of the present invention is simple to operate, and data are comprehensive.
Description
Technical field
The present invention relates to a kind of preparation method of Cardiac cell suspensions, and in particular to a kind of animal hearts single cell suspension
Preparation and detection method.
Background technology
Mouse model experiment is to carry out the important method of scientific research, compared with the animal models such as rat, in inflammation
The inflammatory cell classification in mouse source, cell marking, experiment associated antibodies etc. are more detailed, abundant in the experiment of immunology.
The research of inflammatory cell not only needs qualitative and morphologic research, and it is more accurate, comprehensive that quantitative and functional study can provide
Information.
Lesion is in focal distribution in mouse heart ischemic injuries animal model, and pathological section is vulnerable to materials error interference
And field of view influences.
With the continuous development that heart disease is studied, heart isolated experiment is increasingly valued by the people, especially had
The Cardiomyocytes of standby normal physiological function have turned into research cardiac metabolism, function, the important foundation of pathophysiological mechanism.Such as
Fruit carries out FCM analysis after directly preparing heart individual cells suspension, can not only quantify, and can also apply multispecific antibody mark
Note so that distinguish cell subsets in more detail.
In the organ that the blood sinus such as liver, spleen are abundant, collagenous fibres are few, prepare single cell suspension and be relatively easy to, to internal organs
It is ground, and in this flesh sexual organ of heart, rich in collagenous fibres, how to prepare heart single cell suspension and to cell
Activity influence is minimum, also lacks system, elaborate report.
At present, the method for the detection to cell mass is：Single cell suspension is layered liquid method or Percoll points by Ficoll
Facs analysis aim cell, relative change of the research purpose cell in leucocyte, the party are carried out after layer liquid method enrichment of leukocytes
Method complex operation, and lack the contrast with cardiac muscle cell's sum, more comprehensively data can not be provided.
The content of the invention
It is an object of the invention to provide a kind of preparation of animal hearts single cell suspension and detection method, this method solve
Prior art does not provide specific heart single cell suspension preparation method, and the problem of detection method complex operation, Neng Gouwen
It is fixed, reliably prepare heart single cell suspension, and detection method is simple, can be comprehensively thin in analysis of cardiac single cell suspension
Born of the same parents' subgroup（Cell colony with different cellular antigens）.
In order to achieve the above object, the invention provides a kind of preparation method of animal hearts single cell suspension, this method
Comprising：
Step 1：Make animal hearts ischemia-reperfusion injury model；
Step 2：Animal isolated heart tissue is taken, washes off blood, heart digestive juice is added, shreds, mixes digestion；
Step 3：The mixed solution that filtration step 2 obtains, centrifugation, supernatant is removed, terminate digestion, added phosphate buffer and hang
It is floating, erythrocyte cracked liquid is added, centrifugation, removes supernatant, phosphate buffer is added and suspends, it is unicellular outstanding to obtain animal hearts
Liquid.
In step 2, described heart digestive juice includes：RPMI-1640 culture mediums, Collagenase I and DNase；
The quality of described heart digestive juice：The volume of animal isolated heart tissue is 1.8 ~ 2.2g：11mL.
In step 2, the volume of described RPMI-1640 culture mediums：Collagenase I quality：DNase quality
=1mL：1.6mg：0.2mg.
In step 2, described animal hearts tissue uses phosphate buffer to rinse to remove blood.
In step 2, described digestion is carried out at ambient temperature, is digested 60min and is interrupted mixing.
In step 3, described filtering uses the filter screen of 140 mesh.
In step 3, the speed of described centrifugation is 1000 ~ 1200rpm, and the time of centrifugation is 10min for the first time, second
The time of secondary centrifugation is 5min.
In step 3, the phosphate buffer and erythrocyte cracked liquid added in described mixed solution（It is public purchased from BD
Department, model：349202, specification 100mL）Volume ratio be 1：10.Erythrocyte cracked liquid is used to remove red blood cell and does not destroy the heart
Dirty cell.
Described animal is mouse.
Present invention also offers a kind of detection method of animal hearts single cell suspension, the animal heart of detection method detection
Dirty single cell suspension is obtained by the preparation method of animal hearts single cell suspension as mentioned, and the detection method includes：
Animal hearts single cell suspension is taken, CD45, CD11c and Ly6C antibody or Isotype control is added, marks described animal
Leucocyte in heart single cell suspension, and BMDC and monocyte, phosphate buffer rinse, lucifuge, streaming
Cell detection；Described CD45 antibody or the leucocyte of Isotype control mark are chosen, then analyzes and is marked in CD45 positive cells
CD11c, Ly6C cell subsets are remembered.
The preparation of the animal hearts single cell suspension of the present invention and detection method, solve prior art and do not provide specifically
Heart single cell suspension preparation method, and the problem of detection method complex operation, there is advantages below：
（1）Trypsase is not used in the heart digestive juice of the present invention, to reduce the destruction to cell；
（2）In the heart digestive juice of the present invention in addition to clostridiopetidase A I, DNase is added to reduce cell agglutination；
（3）The heart digestive juice of the present invention can be stablized, reliably vitellophag, and obtained cell content is higher, cell number
About 1 × 106~2×106It is individual；
（4）The detection method of the animal hearts single cell suspension of the present invention first determines to analyze again after CD45 mark leucocytes
The aim cell group of CD11c, Ly6C mark, change of the observation aim cell group in total cardiac muscle cell, simple to operate, data
Comprehensively.
Embodiment
Technical scheme is described further with reference to embodiments.
A kind of preparation method of animal hearts single cell suspension, this method include：
Step 1：Make animal hearts ischemia-reperfusion injury model；
Step 2：Animal isolated heart tissue is taken, washes off blood, heart digestive juice is added, shreds, mixes digestion；Shredding can
Strong operability, the contact area of cardiac muscular tissue and digestive juice can be expanded；
Step 3：The mixed solution that filtration step 2 obtains, centrifugation, supernatant is removed, terminate digestion, added phosphate buffer and hang
It is floating, erythrocyte cracked liquid is added, centrifugation, removes supernatant, phosphate buffer is added and suspends, it is unicellular outstanding to obtain animal hearts
Liquid.
In step 2, heart digestive juice includes：RPMI-1640 culture mediums（Purchased from Hyclone, model
SH30809.01B）, Collagenase I（Clostridiopetidase A I, purchased from Gibco companies, model 17100017）And DNase
（Deoxyribonuclease, deoxyribonuclease）.The DNase is DNase I（Purchased from MBI companies, model EN0521）.
RPMI-1640 culture mediums provide cell suitable liquid environment in vitro, maintain cell function and activity.
Adult mouse heart is flesh sexual organ, rich in collagenous fibres, is not enriched like the internal organs such as liver, spleen blood sinus, collagen
The few organ of fiber is tender, can not be ground and obtain individual cells suspension.
Cardiac muscle digestion is mainly seen in collection suckling mouse（The SD rats of 1-3 days）Heart, separating myocardium cell or into fiber finer
Born of the same parents carry out cell culture and intervention experiment, and the heart digestive juice used mainly contains trypsase, clostridiopetidase A I, to the heart shredded
Dirty tissue block is digested.Wherein, trypsase is mainly used in the protein of break-up tissue interstitial, and effect is strong, has to cell membrane
Stronger destruction.Cardiac muscle cell, the fibroblast ratio in neonate rat heart cell are high, respectively reach 62%, 30%,
Cell total amount is big, even if using trypsin treatment, finally can still obtain enough cells and carry out in vitro culture.It is but immune
No matter inflammatory cell is all extremely low in the heart content of the new life or adulthood of rat or mouse.Therefore, heart of the invention
Trypsase is not used in digestive juice, to reduce the destruction to cell.
In the heart digestive juice of the present invention in addition to clostridiopetidase A I, DNase is added to reduce cell agglutination.The present invention's
Heart digestive juice can be stablized, reliably vitellophag, and obtained cell content is higher.
In step 2, the volume of RPMI-1640 culture mediums：Collagenase I quality：DNase quality=1mL：
1.6mg：0.2mg, the quality of heart digestive juice：The volume of animal isolated heart tissue is 1.8 ~ 2.2g：11mL.In step 2,
Animal hearts tissue uses phosphate buffer（PBS, Phosphate Buffered Solution）Rinse to remove blood.
PBS（PH7.2~7.4）：NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO410mmol/L, KH2PO4
2mmol/L。
In step 2, digest in room temperature（25℃）Under the conditions of carry out, digestion 60min is simultaneously interrupted mixings, digestion 60min energy
Access 1 × 106~2×106Individual individual cells.
In step 3, using the filter screen of 140 mesh, filter screen is the postdigestive cardiac muscular tissue's block of removal, obtains cell for filtering.
In step 3, the speed of centrifugation is 1000 ~ 1200rpm, and the time of centrifugation is 10min for the first time, second of centrifugation
Time be 5min.Preferably, the speed of centrifugation is 1100rpm, can obtain the cell mass relatively to compact, after being easy to and supernatant
Separation.
In step 3, the phosphate buffer and the volume ratio of erythrocyte cracked liquid added in mixed solution is 1：10.
The animal that above-mentioned preparation method uses is mouse.
A kind of detection method of animal hearts single cell suspension, the animal hearts single cell suspension of detection method detection lead to
The preparation method for crossing animal hearts single cell suspension obtains, and the detection method includes：
Animal hearts single cell suspension is taken, CD45, CD11c and Ly6C antibody or Isotype control is added, marks described animal
Leucocyte in heart single cell suspension, and BMDC and monocyte, phosphate buffer rinse, lucifuge, streaming
Cell detection；CD45 antibody or the leucocyte of Isotype control mark are chosen, then analyzes and is marked with CD45 positive cells
CD11c, Ly6C cell subsets.
Experimental group is above-mentioned animal hearts single cell suspension, and positive controls are intact animal heart single cell suspension, can
Prepared using intact animal heart tissue according to the step 2 in the preparation method of the present invention and 3.
Isotype control refers to use and primary antibody（Such as CD45, CD11c and Ly6C antibody）Identical Species origin, identical hypotype,
The immunoglobulin of same dose and identical immune globulin bletilla hypotype, for eliminating because antibody non-specific binding is to carefully
Cellular surface and reasons for its use dye, and the combination for determining primary antibody is specific.
The FCM analysis method of immune organ is because tissue is different, and the FCM analysis method of use is according to specific
Organ-tissue determines.The detection method of the animal hearts single cell suspension of the present invention first determines again to divide after CD45 mark leucocytes
The aim cell group of CD11c, Ly6C mark is analysed, change of the observation aim cell group in total cardiac muscle cell, operation is simpler,
Data more comprehensively, are analyzed simpler clear and definite.
Experimental example 1 makes animal hearts ischemia-reperfusion injury model
The preparation method of animal hearts ischemia-reperfusion injury model is specific as follows：
Step 1 '：Etherization, mouse trachea cannula, lung ventilator is connected, set lung ventilator tidal volume 200-300 μ L, frequency 120
Beat/min, connect electrocardiogram；
Step 2 '：Left front chest is cut, and successively separates hypodermis, exposes surgical field of view with thorax dilator in the 3rd intercostal,
Open pericardium, exposure heart, using left auricle of heart and pulmonary conus for mark searching descending anterior branch, with 8-0 silk threads under left auricle of heart 1-
Inserting needle at 2mm, sham-operation group（Positive controls）Only thread and do not connect bundle, one is made a call to around two circles after ischemia-reperfusion operation group threading
Slip-knot；
Step 3 '：Visually observe ligature to the cardiac muscle at apex of the heart position to lose color, activity weakens, and characteristic ST sections occurs in electrocardiogram
Raise and merged with broadening QRS complex, ligatured successfully；
Step 4 '：Leave and take outside longer slip-knot the end of a thread to chest in case loosen, continue assisted mechanical ventilator and second after suturing the wall of the chest
Ehterization, the slip-knot the end of a thread ligatured outside 30 minutes post-tensioning chests are loosened, and ECG ST section gradually falls after rise, it was demonstrated that myocardial ischemia is again
Perfusion model is successfully prepared.
Animal hearts ischemia-reperfusion injury model can also use conventional method to make；Sham-operation group is as positive controls
Animal model.In sham-operation group and ischemia-reperfusion operation group, every group of 4-7 mouse.
Experimental example 2 prepares animal hearts single cell suspension
Mouse heart is taken in Reperfu- sion 24h, 48h, 72h, 7d respectively, prepares heart single cell suspension, concrete operations are as follows：
（1）Take animal isolated heart tissue about 0.2g, PBS to wash off blood, the digestion of 1mL hearts is added in 2mL centrifuge tubes
Liquid, move in 15mL centrifuge tubes, add 10mL heart digestive juices, room temperature places 60 minutes, and interruption mixes therebetween, obtains mixing molten
Liquid；
（2）Filter mixed solution obtained above with 140 mesh filter screens, collect filtered solution in 15mL centrifuge tubes, 1100rpm from
Centrifuged 10 minutes under heart speed, remove supernatant, added 200uL PBS and suspend, add 2mL erythrocyte cracked liquids, darkroom 5 is divided
Clock, centrifuged 5 minutes under 1100rpm centrifugal speeds, remove supernatant, added 1mL phosphate buffers and suspend, obtain the animal heart
Dirty single cell suspension.
Cell count is carried out using conventional method to the animal hearts single cell suspension of preparation, cell number is about 1 × 106~2
×106It is individual.
The detection method of the animal hearts single cell suspension of experimental example 3
The experiment material used in flow cytometry tests, it is specific as follows：
CD45 antibody is PE-Cy5 anti-mouse CD45（Purchased from eBioscienc companies of the U.S., article No. 15-0451-81）,
Its Isotype control is PE-Cy5 Rat IgG2b Isotype Control（EBioscienc companies of the U.S., article No. 15-4031-
81）；
CD11c antibody is PE anti-mouse CD11c（Purchased from eBioscienc companies of the U.S., article No. 12-0114-81）, its
Isotype control is PE Armenian Hamster IgG Isotype Control（EBioscienc companies of the U.S., article No. 12-
4888-81）；
Ly6C antibody is FITC Rat Anti-Mouse Ly6C（Purchased from U.S. company BD, article No. 553104）, its Isotype control
For FITC Rat IgM, κ Isotype Control（Purchased from U.S. company BD, article No. 553942）；
FACS flow cytometers（Purchased from Becton Dickinson companies）.
CD45 is LCA（Leukocyte common antigen, LCA), on all leucocytes all
There is expression, mark leucocyte.CD11c marks BMDC, Ly6C mark monocytes.
Specific experiment process is as follows：
Take 200 μ L animal hearts single cell suspensions while add 0.3 μ L CD45 antibody or Isotype control, 2.5 μ L CD11c antibody
Or Isotype control and 1 μ L Ly6C antibody or Isotype control, 10 minutes, lucifuge after PBS is washed once, FCM analysis is treated, together
Type control tube is only set in initial test antibody specificity.Leucocyte is marked with CD45, it is further to choose CD45 positive cells
Row is with CD11c, Ly6C mark cell subsets.200000-700000events cell numbers are obtained during cell flow cytometer detection（According to it
Middle CD45 positive cells ratio adjustment, that is, collect at least about 1000 CD45 positive cells）Analyzed, experimental result such as following table
1 and table 2.
The sham-operation group of table 1（sham）And ischemia-reperfusion 24h operation groups（RI 24h）CD45 positive cells ratio and its
The relative scale of four groups of cells of middle CD11c, Ly6C mark（* p ＜ 0.05）
| Packet | Sham | RI 24h |
| CD45 | 0.90±0.25 | 13.88±10.70* |
| Q1（CD11c+Ly6C-） | 25.58±15.70 | 11.73±1.84* |
| Q2（CD11c+Ly6C+） | 6.87±3.32 | 30.53±3.70* |
| Q3（CD11c-Ly6C+） | 56.72±19.90 | 50.03±5.08 |
| Q4（CD11c-Ly6C-） | 10.81±4.23 | 7.72±2.15 |
Note：CD45 represents ratio of the CD45 positive cells in myocardium individual cells suspension；Q1（CD11c+Ly6C-）、Q2
（CD11c+Ly6C+）、Q3（CD11c-Ly6C+）、Q4（CD11c-Ly6C-）It is CD45 positive cells further according to CD11c, Ly6C antibody
Four groups of cells of mark situation division；What Q1, Q2, Q3 and Q4 group counted in table 1 accounts for general cell for each group cell（Q1+ Q2+
Q3+ Q4）Relative scale；* p ＜ 0.05 are represented, relatively have statistically-significant difference with sham-operation group.
The percentage change of each group cells on total cells number of table 2（* p ＜ 0.05）
| Packet | Sham | RI 24h |
| CD45 | 0.90±0.25 | 13.88±10.70* |
| Q1（CD11c+Ly6C-） | 0.243±0.174 | 1.63±1.23* |
| Q2（CD11c+Ly6C+） | 0.062±0.032 | 4.18±3.11* |
| Q3（CD11c-Ly6C+） | 0.498±0.16 | 7.06±5.95* |
| Q4（CD11c-Ly6C-） | 0.096±0.039 | 1.01±0.69* |
Note：Total cell number is the TCS in myocardium individual cells suspension.
Through above-mentioned cell flow cytometer showed, as can be seen from Table 1 and Table 2, myocardial ischemia-reperfusion injury（RI,
Reperfusion injury）24h hearts CD45 positive cells significantly raise afterwards, the BMDC of CD11c marks and Ly6C
Each cell subset such as the monocyte of mark also significantly raises, and wherein double positive cells rise is the most prominent.
In summary, the preparation of animal hearts single cell suspension of the invention and detection method can be carried out to heart cell
Quantitative study, and the changing of cell phenotype, intercellular mutually conversion can be studied by different label.
Although present disclosure is discussed in detail by above preferred embodiment, but it should be appreciated that above-mentioned
Description is not considered as limitation of the present invention.After those skilled in the art have read the above, for the present invention's
A variety of modifications and substitutions all will be apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Claims (10)
1. a kind of preparation method of animal hearts single cell suspension, it is characterised in that this method includes：
Step 1：Make animal hearts ischemia-reperfusion injury model；
Step 2：Animal isolated heart tissue is taken, washes off blood, heart digestive juice is added, shreds, mixes digestion；
Step 3：The mixed solution that filtration step 2 obtains, centrifugation, supernatant is removed, terminate digestion, added phosphate buffer and hang
It is floating, erythrocyte cracked liquid is added, centrifugation, removes supernatant, phosphate buffer is added and suspends, it is unicellular outstanding to obtain animal hearts
Liquid.
2. the preparation method of animal hearts single cell suspension according to claim 1, it is characterised in that in step 2, institute
The heart digestive juice stated includes：RPMI-1640 culture mediums, Collagenase I and DNase；
The quality of described heart digestive juice：The volume of animal isolated heart tissue is 1.8 ~ 2.2g：11mL.
3. the preparation method of animal hearts single cell suspension according to claim 2, it is characterised in that in step 2, institute
The volume for the RPMI-1640 culture mediums stated：Collagenase I quality：DNase quality=1mL：1.6mg：0.2mg.
4. the preparation method of animal hearts single cell suspension according to claim 3, it is characterised in that in step 2, institute
The animal hearts tissue stated uses phosphate buffer to rinse to remove blood.
5. the preparation method of the animal hearts single cell suspension according to any one in claim 1-4, it is characterised in that
In step 2, described digestion is carried out at ambient temperature, is digested 60min and is interrupted mixing.
6. the preparation method of animal hearts single cell suspension according to claim 5, it is characterised in that in step 3, institute
The filtering stated uses the filter screen of 140 mesh.
7. the preparation method of animal hearts single cell suspension according to claim 6, it is characterised in that in step 3, institute
The speed for the centrifugation stated is 1000 ~ 1200rpm, and the time of centrifugation is 10min for the first time, and the time of second of centrifugation is 5min.
8. the preparation method of the animal hearts single cell suspension according to claim 6 or 7, it is characterised in that in step 3
In, the phosphate buffer and the volume ratio of erythrocyte cracked liquid that are added in described mixed solution are 1：10.
9. the preparation method of animal hearts single cell suspension according to claim 8, it is characterised in that described animal is
Mouse.
A kind of 10. detection method of animal hearts single cell suspension, it is characterised in that the animal hearts list of detection method detection
Cell suspension is obtained by the preparation method of the animal hearts single cell suspension as described in any one in claim 1-9, should
Detection method includes：
Animal hearts single cell suspension is taken, CD45, CD11c and Ly6C antibody or Isotype control is added, marks described animal
Leucocyte in heart single cell suspension, and BMDC and monocyte, phosphate buffer rinse, lucifuge, streaming
Cell detection；
Described CD45 antibody or the leucocyte of Isotype control mark are chosen, then analyzes and is marked with CD45 positive cells
CD11c, Ly6C cell subsets.
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