CN108279145A - Periphery red blood cell lysis method in the analysis of tree shrew Immunophenotyping - Google Patents
Periphery red blood cell lysis method in the analysis of tree shrew Immunophenotyping Download PDFInfo
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- 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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- 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—Electro-optical investigation, e.g. flow cytometers
- G01N15/1404—Fluid conditioning in flow cytometers, e.g. flow cells; Supply; Control of flow
Abstract
The present invention relates to tree shrew peripheral blood (PB) erythrocyte splitting methods.It passes through BD Pharm LyseTMTree shrew whole blood is cracked twice, and cracking condition, that is, pyrolysis time, cracking number and the volume ratio of lysate and whole blood are optimized, the erythrocyte splitting scheme for tree shrew peripheral blood cells Immunophenotype analysis for establishing optimization, the assessment for the further immune system and immune response of research tree shrew experimental animal provide important technology platform.
Description
Technical field
The present invention relates to biological immune related fields, and in particular to peripheral red blood cells cleavage method is more particularly to set
Periphery red blood cell lysis method in the analysis of Shrew Immunophenotypings.
Background technology
Tree shrew is a kind of emerging experimental animal[1,2], in the research of animal model of human disease, increasingly receive
The attention of scientists, including spreading venereal diseases[3], metabolic disease[4], neurological disease[5]And cancer[6].However, due to lacking business
The reagent of change so that we are very limited to the cell phenotype understanding of tree shrew, these hinder tree shrew opening as experimental animal
Hair application.In clinical and Research of Animal Model for Study, flow cytometry is research host to pathogen or antigen-reactive, hematopoiesis
One of the basic skills of systemic disease, immune correlated disease and stem cell etc..Erythrocyte splitting is peripheral blood flow cytometry
In an important link, although it is not essential for some antibody in clinical analysis, after splitting erythrocyte
It analyzes it again and not only saves antibody, and the Immunophenotype analysis result of human peripheral blood is also very crucial[7].
In this research, we have rated the effect that is cracked to tree shrew peripheral red blood cells of reagent of four kinds of commercializations or document report, and
Erythrocyte splitting method and condition is optimized, obtains best tree shrew peripheral red blood cells cleavage method, and pass through
Flow cytometry immunophenotype is verified.
Although reagent of many for erythrocyte splitting can be bought on the market, they pass through different mechanism
It plays a role, and shows different erythrocyte splitting effects[8].In addition, being cracked currently without about tree shrew peripheral red blood cells
The report of reagent, and not yet determine the best approach of tree shrew erythrocyte splitting.
Bibliography
1 Yao YG. Creating animal models, why not use the Chinese tree shrew
(Tupaia belangeri chinensis)Zool Res 2017, 38: 118-126
2 Xiao J, Liu R, Chen CS. Tree shrew (Tupaia belangeri) as a novel
laboratory disease animal model. Zool Res 2017, 38: 127-137
3 Zi-feng Yang JZ, Yu-tong Zhu, Yu-tao Wang, Rong Liu, Sui-shan Zhao,
Run-feng Li, Chun-guang Yang, Ji-qiang Li and Nan-shan Zhong. The tree shrew
provides a useful alternative model for the study of influenza H1N1 virus.
2013, Virology Journal: 1-9
4 Wu X, Xu H, Zhang Z, Chang Q, Liao S, Zhang L, Li Y, et al.
Transcriptome Profiles Using Next-Generation Sequencing Reveal Liver Changes
in the Early Stage of Diabetes in Tree Shrew (Tupaia belangeri chinensis). J
Diabetes Res 2016, 2016: 6238526
5 Sonnay S, Poirot J, Just N, Clerc AC, Gruetter R, Rainer G, Duarte JMN.
Astrocytic and neuronal oxidative metabolism are coupled to the rate of
glutamate-glutamine cycle in the tree shrew visual cortex. Glia 2017
6 Ye L, He M, Huang Y, Zhao G, Lei Y, Zhou Y, Chen X. Tree shrew as a new
animal model for the study of lung cancer. Oncology letters 2016, 11: 2091-
2095
7 Einwallner E, Subasic A, Strasser A, Augustin D, Thalhammer R, Steiner
I, Schwarzinger I. Lysis matters: red cell lysis with FACS Lyse affects the
flow cytometric enumeration of circulating leukemic blasts. Journal of
immunological methods 2013, 390: 127-132
8 Bossuyt X, Marti GE, Fleisher TA. Comparative analysis of whole blood
lysis methods for flow cytometry. Cytometry 1997, 30: 124-133
9 Pan Q, Ye L, Deng Z, Li L, Liu H. Effects of red blood cell lysing
solutions on the detection of peripheral basophils of healthy normals and SLE
patients by flow cytometry. J Immunoassay Immunochem 2014, 35: 368-377
10 Desmeules P, Dufour M, Fernandes MJ. A rapid flow cytometry assay for
the assessment of calcium mobilization in human neutrophils in a small volume
of lysed whole-blood. Journal of immunological methods 2009, 340: 154-157
11 Stabel TJ, Bolin SR, Pesch BA, Rahner TE. A simple and rapid flow
cytometric method for detection of porcine cell surface markers. Journal of
immunological methods 2000, 245: 147-152。
Invention content
The present invention evaluates and compares four kinds of reagents and validity that scheme cracks tree shrew peripheral red blood cells, selects
Most effective lytic reagent carries out the optimization of cracking condition, to obtain most effective tree shrew peripheral red blood cells cleavage method.
Technical solution provided by the invention is：A kind of tree shrew peripheral red blood cells cleavage method, this method include following step
Suddenly：
1）Whole blood is acquired from tree shrew tail vein；
2）By the 1x BD Pharm Lyse of 8-12 times of volumeTMIt is added in tree shrew whole blood；
3）Soft vortex mixing is carried out immediately；
4）It is protected from light incubation 10-20 minutes at room temperature；
5）Room temperature is centrifuged in 180-220, centrifugation time 5 minutes；
6）Supernatant is sucked, does not destroy precipitation；
7）Cell precipitation is resuspended with the dye solution of initial whole blood same volume；
8）The 1x BD Pharm Lyse of the above cell suspension 8-12 volumes are addedTM；
9）Soft vortex mixing is carried out immediately；
10）Room temperature is protected from light incubation 3-7 minutes；
11）Room temperature is centrifuged in 180-220, centrifugation time 5 minutes；
12）Supernatant is sucked, precipitation is not broken up；
13）It is spare that precipitation is resuspended with dye solution.
Preferably, the dye solution, group are divided into containing 0.1%BSA and 0.01%NaN3PBS.
Preferably, the centrifugation of the 5th step or the 10th step centrifuges for 203g, centrifugation time 5 minutes.
Preferably, the 4th step be protected from light at room temperature incubation be 15 minutes.
Preferably, the 10th step be protected from light at room temperature incubation be 5 minutes.
Preferably, the 2nd step is by the 1x BD Pharm Lyse of 10 times of volumesTMIt is added to containing in tree shrew whole blood.
Preferably, the BD Pharm LyseTMSpecific number is Cat#:555899.
The present invention establishes the erythrocyte splitting scheme for the analysis of tree shrew PB Immunophenotypings of an optimization, can
It is effectively removed red blood cell, and can ensure the activity of peripheral blood mononuclear cells to the maximum extent, and is completely suitable for tree shrew periphery
The flow cytometry Immunophenotyping of blood, for further the research immune system of tree shrew experimental animal and commenting for immune response
Estimate and provides important technology platform.
Description of the drawings
Tetra- kinds of Fig. 1 are different, and tree shrew peripheral red blood cells lytic effect is compared in cracking, wherein (A) is according to primate
Peripheral blood flow cytometry after the erythrocyte splitting drawn with muroid data（FSC/SSC）Pattern usually has 5 types of populations：It is small
Particle（Red blood cell, fragment and blood platelet）, lymphocyte, myeloid cell, granulocyte and PI positive cells；(B) tree shrew peripheral blood
Flow cytometry after erythrocyte splitting（FSC/SSC）.50ul anti-freezing tree shrew whole bloods are transferred in streaming pipe, into anticoagulation
Erythrocyte cracked liquid (a, BD Pharm Lyse the Lysing Buffer of the volume of manufacturer's recommended or document report are added; b,
Distilled water; c, BioLegend RBCs Lysis/Fixation Buffer; d, 1.5% formal
Dehydrate after), erythrocyte splitting is carried out according to the condition recommended or reported, passes through Flowjo 10.0.7 softwares and carries out data
Analysis（FSC/SSC）.
Fig. 2 tree shrew peripheral red blood cells cracking conditions optimize.The tree shrew whole blood of 50ul anti-freezings is transferred to streaming pipe, such as schemes
It is shown respectively to pyrolysis time（15 minutes, 20 minutes and 25 minutes）, cracking number and lysate and volume of whole blood ratio（10：1 He
20：1）Analyzed and compared its influence to erythrocyte splitting effect.Wherein, (A) passes through flow cytometer and Flowjo
10.0.7 software is analyzed（FSC/SSC）Physical property after erythrocyte splitting.(B) pass through flow cytometer and Flowjo
10.0.7 software is analyzed（FSC/SSC）PI positive cells after erythrocyte splitting are horizontal.(A) it is at least repeated 10 times with (B).
(C) influence of the tree shrew peripheral blood initial volume to erythrocyte splitting effect.The initial tree of 50-400ul different volumes as shown in the figure
Shrew volume of whole blood is transferred to streaming pipe respectively, the condition optimized according to front（It cracks 10 minutes for the first time, second of cracking 5
Minute and the volume ratio of lysate and whole blood are 10：1）Erythrocyte splitting is carried out, flow cytometer and Flowjo are passed through
10.0.7 remaining little particle and PI are positive in PBMCs after software analysis (SSC vs FSC and SSC vs PI) erythrocyte splitting
Property cell rate.
The erythrocyte splitting method of Fig. 3 optimizations is for CD4 positive cells in parsing tree Shrew peripheral bloods.Pass through the item of optimization
It is anti-with 2 of the clone OX-35 for CD4 surface markers respectively after 300 ul tree shrew peripheral bloods of part pair carry out erythrocyte splitting
Body（Respectively from BD and Biolegend companies）It is dyed, it is then soft by BD FACSCalibur and Flowjo 10.0.7
Part is analyzed.Cell is analyzed by FSC/SSC first.After non-erythrocytic granule gating（Left-hand column）, further pass through
FL1 and FL2 multichannel analysis, after the control of antibody not being added to iris out the cell of autofluorescence（Middle column）, finally carry out CD4
The analysis of positive cell（Right-hand column）, which is repeated 3 times.
Specific implementation mode
One, research process
1, experimental animal
The F1 generation bearing tree Shrew that this research uses（1-2 Sui）, come from China Medical Sciences Academy Medical Biology Institute's tree shrew kind
Matter resource center, experimental animal production licence SCXK (Yunnan) K2013-0001, uses licensing SYXK (Yunnan) K2013-
0001.All animals using program all by from Medical Biology experimental animal Ethics Committee of the Chinese Academy of Medical Sciences examine and
Approval.
, experiment reagent
Four kinds of red blood cell lysing agents：BD Pharm LyseTM（BD, Cat#:555899）, BioLegend RBCs Lysis/
Fixation solution（BioLegend, Cat#: 422401）, 1.5% formalin and distilled water.Antibody：BD companies
PE- anti-CD 4 antibodies（Clone：OX-35, Cat#: 554838）, the PE- anti-CD 4 antibodies of Biolegend companies（Clone：OX-35,
Cat#: 100411）.Other chemical reagent：PBS（Amresco, Cat#：E404-200TABS）, BSA（SIGMA, Cat#：
A3912）, 10xPBS（Solarbio, Cat#：P1022-500）;Propidium iodide（PI）（Invitrogen, Cat#：P3566）.Dye
Color buffer solution：Contain 0.1%BSA and 0.01%NaN31x PBS.
3, tree shrew peripheral blood acquires
Caudal puncture is taken a blood sample.First by the outside of belly cropping of tree shrew tail portion, then sterilized with 70% alcohol wipe, and gently beating makes tree
Shrew tail veins are full.With 1ml syringes blood is extracted close to parallel piercing tree shrew tail epigastric vein.0.5-1.0ml is collected every time
Blood, is transferred in the pipe that 5ml contains anti-coagulant heparin and quick mixing.
4, the validity of flow cytometry assessment tree shrew erythrocyte splitting
Purpose of the present invention is to find tree shrew Immunophenotyping analysis preferred plan, therefore we by flow cytometry come
The effect of decision tree Shrew erythrocyte splittings.Pass through sample after BD FACSCalibur flow cytometry analysis tree shrew erythrocyte splittings
Product, 30000 cells of each sample collection, and pass through the analysis of Flowjo 10.0.7 softwares progress data.First, pass through FSC
/ SSC analyzes to assess the cell mass of the PBMC after erythrocyte splitting, according to analytical model（Fig. 1 a）, 3 differences should be shown
Cell mass：Smaller lymphocyte populations, medium sized myeloid cell group and in addition to little particle（Red blood cell, fragment and blood are small
Plate）Except maximum granulocyte.Then the survival rate of PBMC after erythrocyte splitting is assessed by PI dyeing.Finally, to each
Reagent or erythrocyte splitting method have carried out net assessment, and determine prioritization scheme.These guilding principles are for comparing four kinds of examinations
The validity of agent splitting erythrocyte, and optimize erythrocyte splitting scheme.
5, tree shrew peripheral red blood cells crack
(1)BD Pharm LyseTMCracking to tree shrew red blood cell
Erythrocyte splitting step is according to BD Pharm LyseTMProducts instruction be modified slightly.By the 1x of 10 times of volumes
BD Pharm LyseTMIt is added in the tree shrew whole blood of 1 volume, then carries out slight vortex mixing.It is protected from light and cracks 15 at room temperature
After minute, mixture is centrifuged 5 minutes with the speed of 203g at room temperature.After removing supernatant, with 1ml PBS-BSA（0.1%
BSA and 0.1%NaN3）Suspension cell precipitates, and is centrifuged 5 minutes with 203g speed at room temperature.Finally with 300 μ l dye solution weights
Outstanding cell precipitation is used for flow cytometry.
(2) cracking of the BioLegend RBCs Lysis/Fixation solution to tree shrew red blood cell
Erythrocyte splitting step is according to the products instruction of BioLegend RBCs Lysis/Fixation solution
It is modified slightly.BD Pharm Lyse are used with above-mentionedTMCracking scheme it is almost the same, in addition to the volume ratio of lysate and whole blood
Example is different, i.e.,：The ratio of BioLegend RBCs Lysis/Fixation solution is 20：1, and BD Pharm
LyseTMIt is 10：1.
Cracking of (3) 1.5% formaldehyde to tree shrew red blood cell
The method that the program cracks swine erythrocyte with reference to Stabel TJ.First, 40 bodies are added in the tree shrew whole blood of 1 volume
Then 1.5% long-pending formaldehyde carries out slight vortex mixing.After being incubated 30 minutes at room temperature, by mixture at room temperature with 203g
Speed centrifuge 5 minutes.After removing supernatant, cell precipitation is resuspended with 300 μ l dye solutions and is used for flow cytometry.
(4) cracking of the distilled water to tree shrew red blood cell
The program is according to the document reported[2,3]（Desmeules P etc., Pan Q etc., aforementioned reference is seen in specific source）
It modifies.50ul tree shrew whole bloods are added in the healthy and free from worry centrifuge tubes of 15ml, with 10ml pipette, extract 9ml distilled water and tree shrew
Whole blood softly mixes 2-3 times, 10 x PBS of 1ml is added immediately, and overturn mixing rapidly.Mixture is passed through into 100- μm of cell
Filter filters and centrifuges 5min with 203g speed.Supernatant is removed, cell precipitation, which is resuspended, with 300 μ l dye solutions is used for
Flow cytometry.
6, the optimization of erythrocyte splitting condition
In above-mentioned four kinds of reagents, most effective lytic reagent is selected to carry out the optimization of cracking condition, it is most effective to obtain
Tree shrew peripheral red blood cells crack scheme.The present invention tests and compares to following erythrocyte splitting condition：Pyrolysis time is split
Solve the volume ratio of number, lysate and whole blood.Cleavage method is as follows twice：1）The total time of cracking scheme and primary cracking twice
The total time of scheme is identical, including the time of first time erythrocyte splitting and 5 minutes time of second of erythrocyte splitting.2）One
The pyrolysis time of secondary cracking scheme is as first time pyrolysis time.3）After first time erythrocyte splitting, 5 points are centrifuged with 203g speed
Clock.Then the lysate for being suspended and being precipitated with 50 μ l dye solutions, and 10 times of volumes are added carries out second and cracks, the time 5
Minute.Other conditions are identical with Products Show or document report.
7, immunophenotype dyes
According to products instruction, by the PE- anti-CD 4 antibodies of 1 μ l（BD or Biolegend）It is added to 100 μ l（1×106It is a
Cell）In cell suspending liquid after tree shrew erythrocyte splitting, then mild mixing is protected from light incubation 20 minutes in 4 DEG C.Use 2ml
Dye solution wash cell under the following conditions twice：It is centrifuged 5 minutes with 203g speed at room temperature.Supernatant is removed, is used
300 μ l dye solutions are resuspended cell precipitation and carry out flow cytometry, and 30,000 cell of each sample collection is for analyzing.
8, propidium iodide stain
In order to assess the cell viability after tree shrew erythrocyte splitting, before flow cytometry, to 100ul cell suspensions（1×
10 6A cell）1 μ l propidium iodides of middle addition（PI）（1.0mg / ml）.With FL2 multichannel analysis propidium iodide positive cells
Rate.Cell viability is calculated by propidium iodide exclusion（Aforementioned reference is seen in Liu H etc., specific source）.
Two, result of study is analyzed
First, according to the peripheral blood data of primate after erythrocyte splitting or mouse, a flow cytometry is established
（FSC vs SSC）Model.The model shows that peripheral blood is generally divided into four clearly cell masses after erythrocyte splitting, i.e.,：Leaching
Bar cell, myeloid cell, granulocyte and PIhighCell mass little particle（Including：Red blood cell and cell fragment etc.）（Fig. 1 a）.It is logical
This analysis model is crossed, we pick four kinds of red blood cell lysing agents available on the market and method（BD Pharm LyseTM
（BD, Cat#:555899）, BioLegend RBCs Lysis/Fixation solution（BioLegend, Cat#:
422401）, 1.5% formalin and distilled water）, tree shrew peripheral red blood cells are cracked, then their effect is carried out
Flow cytometry and compare.The result shows that the tree shrew peripheral blood after erythrocyte splitting（PB）With primate or muroid
Peripheral blood（PB）Pattern is similar.Further comparative analysis is found, in the four kinds of reagents tested in our current research, is widely used in clinic
With BD Pharm Lyse in laboratory researchTMIt shows best lytic effect, passes through FSC and SSC（Fig. 1 a and Fig. 1 b）Point
Analysis shows 3 kinds of different leukocyte populations, although still there is a certain number of little particles（Fig. 1 b）.With other three kinds of reagent phases
Than although showing best lytic effect using the distilled water of Hyposmolality lytic cell（Fig. 1 b）, but distill water-splitting
Peripheral blood mononuclear cells afterwards（PBMCs）Show other cell masses spread（Fig. 1 b）, these cells of PI dyeing confirmations can
It can be dead cell（Data are not shown）, show that distilled water destroys some PBMCs [9,10] during erythrocyte splitting.
BioLegend RBCs Lysis/Fixation solution and 1.5% formalin cannot be effectively removed red blood cell, also not
It can obtain clearly PBMCs cell masses（Fig. 1 b）.
Because with BD Pharm LyseTMSplitting erythrocyte can generate apparent and independent PBMC cells in tree shrew PB
Group（Fig. 1 b）, so we select the lysate to carry out subsequent experimental.Nevertheless, BD Pharm LyseTMIt is used according to product
After specification cracks tree shrew peripheral blood, still there is a large amount of red blood cell to remain.May be because of BD Pharm LyseTMIt is only wide
General test is not necessarily suited for the peripheral blood of tree shrew in the peripheral blood of people and mouse.Therefore, next to the condition of the lytic reagent
It is optimized, it is intended to the best approach to tree shrew PB erythrocyte splittings is found, including：Pyrolysis time cracks number and splits
Solve the volume ratio of liquid and whole blood（Fig. 2）.The purpose of these optimizations is to improve the validity of erythrocyte splitting to the maximum extent, and make
It is dead（PI is positive）The percentage of cell is minimum.These optimizations have at least carried out 10 experiments.Finally, it has been found that crack twice
Compared with primary cracking, more red blood cells can be removed, although PI after cracking twice+The percentage of cell is slightly above a secondary fissure
Solution（Fig. 2）.Although the volume ratio of lysate and whole blood does not influence the lytic effect of red blood cell, large volume of lysate（20：
1）Lead to the cell mass for dispersion occurred（Fig. 2 a）, dead cell is confirmed that it is by PI dyeing and flow cytometry（Figure
2b）.In short, cracking for the first time 10 minutes and second cracking 5 minutes and lysate and whole blood 10：The condition of 1 volume ratio,
Best result is shown in the erythrocyte splitting condition tested at six kinds.
By the tree shrew peripheral red blood cells cleavage method of above-mentioned optimization, the initial volume of whole blood has further been inquired into red
The influence of cell cracking effect.The different external whole bloods of 50-400ul are cracked respectively using above-mentioned cleavage method, and little particle is used in combination
Percentage and the survival rate of PBMC assess the validity of erythrocyte splitting.Flow cytometric analysis results are shown, with red thin
The increase of the whole blood initial volume of cellular lysate, little particle are reduced（Fig. 2 c）.In addition, being enhanced using larger whole blood initial volume
The survival rate of PBMC.The whole blood of 300ul and 400ul shows the effect of similar erythrocyte splitting（Fig. 2 C）.These tables of data
Bright, larger whole blood initial volume is conducive to the erythrocyte splitting in tree shrew PB.
To determine whether the PBMCs after erythrocyte splitting is suitable for tree shrew Immunophenotype analysis, we split in 300ul whole bloods
In the PBMC that solution obtains, the two kinds of anti-CD 4 antibodies bought respectively with BD and Biolegend Liang Ge companies dye it.Stream
Formula cell analysis shows that tree shrew PB has the apparent CD4 positive colonies of a group in the PBMC after erythrocyte splitting, point
It Wei 13.7% and 14.5%（Fig. 3）.These results indicate that carrying out the tree shrew that erythrocyte splitting obtains using the scheme of optimization
The quality of PBMC is sufficiently used for the Immunophenotype analysis of tree shrew.
Claims (7)
1. a kind of tree shrew peripheral red blood cells cleavage method, it is characterised in that：Include the following steps：
Whole blood is acquired from tree shrew tail vein；
2）By the 1x BD Pharm Lyse of 8-12 times of volumeTMIt is added in tree shrew whole blood；
3）Soft vortex mixing is carried out immediately；
4）It is protected from light incubation 10-20 minutes at room temperature；
5）Room temperature is centrifuged in 180-220, centrifugation time 5 minutes；
6）Supernatant is sucked, does not destroy precipitation；
7）Cell precipitation is resuspended with the dye solution of initial whole blood same volume；
8）The 1x BD Pharm Lyse of the above cell suspension 8-12 volumes are addedTM；
9）Soft vortex mixing is carried out immediately；
10）Room temperature is protected from light incubation 3-7 minutes；
11）Room temperature is centrifuged in 180-220, centrifugation time 5 minutes；
12）Supernatant is sucked, precipitation is not broken up；
13）It is spare that precipitation is resuspended with dye solution.
2. tree shrew peripheral red blood cells cleavage method as described in claim 1, it is characterised in that：The dye solution,
Its group is divided into containing 0.1%BSA and 0.01%NaN3PBS.
3. tree shrew peripheral red blood cells cleavage method as described in claim 1, it is characterised in that：5th step or the 10th step from
The heart centrifuges for 203g, centrifugation time 5 minutes.
4. tree shrew peripheral red blood cells cleavage method as described in claim 1, it is characterised in that：4th step is to keep away at room temperature
It is 15 minutes that light, which is incubated,.
5. tree shrew peripheral red blood cells cleavage method as described in claim 1, it is characterised in that：10th step is to keep away at room temperature
It is 5 minutes that light, which is incubated,.
6. tree shrew peripheral red blood cells cleavage method as described in claim 1, it is characterised in that：2nd step is by 10 times of volumes
1x BD Pharm LyseTMIt is added to containing in tree shrew whole blood.
7. tree shrew peripheral red blood cells cleavage method as described in claim 1, it is characterised in that：The BD Pharm
LyseTMSpecific number is Cat#:555899.
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|---|---|---|---|---|
| CN113604431A (en) * | 2021-09-02 | 2021-11-05 | 广西医科大学第一附属医院 | Separation method of tree shrew peripheral blood mononuclear cells |
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| US5155044A (en) * | 1987-03-13 | 1992-10-13 | Coulter Electronics, Inc. | Lysing reagent system for isolation, identification and/or analysis of leukocytes from whole blood samples |
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| CN113604431A (en) * | 2021-09-02 | 2021-11-05 | 广西医科大学第一附属医院 | Separation method of tree shrew peripheral blood mononuclear cells |
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