CN107063982A - The method of Flow cytometry chicken T lymphocyte subsets - Google Patents
The method of Flow cytometry chicken T lymphocyte subsets Download PDFInfo
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- 210000000662 T-lymphocyte subset Anatomy 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000000684 flow cytometry Methods 0.000 title claims abstract description 25
- 210000004027 cell Anatomy 0.000 claims abstract description 70
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 47
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 230000002093 peripheral effect Effects 0.000 claims abstract description 25
- 238000000926 separation method Methods 0.000 claims abstract description 24
- 239000007788 liquid Substances 0.000 claims abstract description 23
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- 238000012360 testing method Methods 0.000 claims abstract description 20
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- 230000010100 anticoagulation Effects 0.000 claims abstract description 16
- 238000004043 dyeing Methods 0.000 claims abstract description 14
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- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
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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
- G01N15/1425—Optical investigation techniques, e.g. flow cytometry using an analyser being characterised by its control arrangement
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Abstract
The invention discloses a kind of method of Flow cytometry chicken T lymphocyte subsets, comprise the following steps：Chicken peripheric venous blood is taken, anticoagulation is prepared, is handled with lymphocyte separation medium and obtains peripheral white blood cells；With PBS liquid to the peripheral white blood cells centrifuge washing after, prepare single cell suspension；The single cell suspension is drawn, anti-chicken CD3, CD4, CD8 monoclonal antibody is separately added into, vortex is mixed, in 4 DEG C of lucifuge dyeing；After dyeing, washed with PBS liquid and cell is resuspended, detected using flow cytometer；Testing result is analyzed, chicken T lymphocyte subsets ratio is obtained.The method of Flow cytometry chicken T lymphocyte subsets of the present invention, the difference of processing mode during for Sample pretreatment, detection when regulation, the interpretation of result of voltage and fluorescence compensation causes the problem of testing result fluctuation is big, unstable, establishes a kind of detection method of the chicken T lymphocyte subsets of stable specification.
Description
Technical field
The invention belongs to Cell Measurement Technique field, specifically, it is related to a kind of Flow cytometry chicken peripheral blood T and drenches
The method of bar cell subsets.
Background technology
Flow cytometry (Flow Cytometry, FCM) is a kind of individual cells surface or intracellular chemistry composition to be entered
Row quantitative analysis and the new technology of sorting.FCM mainly will be thin by gathering forward scattering light (FSC) and side scattered light (SSC)
Born of the same parents' size cell different with intracellular granularity is separated.And by recognizing cell surface and intracellular special fluorescence labeling, it is real
Now to the detection of DNA, RNA, cell factor, cell surface antigen etc..The technology is as a kind of rapid cellular analytical technology, not only
Applied to basic medical researches such as immunology, microbiologies, broader applications are in clinical detections such as oncology, hematologies.
Flow cytometry can be special by recognizing cell surface Immunofluorescent signals, come realize t lymphocyte subset group
Detection, contribute to evaluate body cellular immune function.Further, it is also possible to evaluate monoclonal antibody using flow cytometry
Specificity, selected in monoclonal antibody preparation optimal marking signal (such as when detecting chicken periphery blood T lymphocyte,
PE marks have wider array of detection range than the FITC CD3 marked).Found in clinical and scientific research, Sample pretreatment, detection
When voltage and fluorescence compensation regulation, interpretation of result when processing mode can all influence testing result to a certain extent, cause
Detected value fluctuation range is big, testing result is unstable.It there is no at present sub- using flow cytomery chicken periphery blood T lymphocyte
The reliable reference method of group.
The content of the invention
In view of this, the present invention for Sample pretreatment, detection when voltage and fluorescence compensation regulation, interpretation of result when
The difference of processing mode causes the problem of testing result fluctuation is big, unstable, and there is provided a kind of Flow cytometry chicken periphery
The method of blood t lymphocyte subset group, establishes a kind of detection method of the chicken T lymphocyte subsets of stable specification.
In order to solve the above-mentioned technical problem, the invention discloses a kind of Flow cytometry chicken periphery blood T lymphocyte
The method of subgroup, comprises the following steps：
Step 1, chicken peripheric venous blood is taken, anticoagulation is prepared, is handled with lymphocyte separation medium and obtains peripheral white blood cells；
Step 2, with PBS liquid to the peripheral white blood cells centrifuge washing after, prepare single cell suspension；
Step 3, draw in described two parts to two streaming pipes of single cell suspension, portion adds CD3, CD4, CD8 of anti-chicken
Each part of monoclonal antibody, vortex is mixed, in 4 DEG C of lucifuge dyeing, as detection pipe；Another is used as negative set and managed.
Step 4, after dyeing, washed with PBS liquid and cell is resuspended, detected using flow cytometer；
Step 5, testing result is analyzed, chicken T lymphocyte subsets ratio is obtained.
Further, described handled with lymphocyte separation medium obtains peripheral white blood cells, is specially：Pressed in streaming pipe
According to volume ratio 1:1 successively adds lymphocyte separation medium and anticoagulation, it is ensured that anticoagulation and lymphocyte separation medium boundary are clear,
Peripheral white blood cells are obtained after centrifugation.
Further, the rotating speed of the centrifugation is 2500r/min, and the time is 15min.
Further, the single cell suspension for preparing is specially：Weight after peripheral white blood cells are washed with the PBS liquid of precooling
It is outstanding, it is adjusted to cell concentration 1 × 106-1×107Individual/mL single cell suspension, is saved backup in 4 DEG C.
Further, the negative processing mode for setting pipe has two kinds, and one is to use Isotype control reagent dyeing；Two be not contaminate
Color.
Further, the system of selection of Isotype control reagent is specially：The primary antibody of selection surface marker corresponding with antibody is complete
The antibody of exactly the same Species origin, identical hypotype and fluorescence labeling.For example, detection pipe uses the CD4 FITC of the anti-chicken of mouse
It is Mouse IgG1 that its composition is shown on the antibody of mark, specification, then Isotype control is exactly the Mouse of FITC marks
IgG1.It is familiar with after point public sentiment condition of each monoclonal antibody positive cell, negative control is set usually using blank control.
Further, the time of the dyeing is 30min.
Further, the use flow cytometer is detected, including：In the double ginsengs of forward scattering light/side scattered light
Voltage and current linear gain parameter is adjusted in number figure, lymphocyte is separated with peripheral cell；Choose lymphocyte and gating；
Set pipe to set negative area with feminine gender, compensated with detection pipe regulation fluorescence.
Further, the regulation voltage and current linear gain parameter, be specially：Adjust the electricity of lateral scattering optical channel
Pressure, lymphocyte populations are separated with peripheral cell；The current gain of forward scattering optical channel is adjusted, makes cell mass and cell fragment
Separate.
Further, the negative area of the setting is specially：Take negative set ten in pipe flow cytometer, setting scatter diagram
The region of word door lower left is negative area, and regulation voltage makes negative area's inner cell percentage be more than 98%.
Further, the analysis testing result, including：Circle takes target cell and gating, respectively in CD3/CD4, CD3/
Double-negative area, single negative area and double hot spots are accurately defined in CD8, CD4/CD8 quadrantal diagram, CD3 is read+、CD3+CD4+And CD3+
CD8+T cent lymphocytes, so as to obtain chicken T lymphocyte subsets ratio.
Further, the circle takes target cell and gating requires that circle takes the cell mass close to forward scattering optical axis.
Compared with prior art, the present invention can be obtained including following technique effect：
(1) present invention has been successfully established the side of the Flow cytometry chicken T lymphocyte subsets of stable specification
Method, by the target cell during sample pre-treatments, detection and analysis is chosen, compensation adjustment and parameter are selected the problem of enter
Row is improved, it is to avoid the error that human factor is caused in process of the test, improves the authenticity and objectivity of result judgement.
(2) this method is simple and easy to apply, can be widely applied to evaluate infection communicable disease, metabolic in poultry production practice
The cellular immune function state of chicken when disease and nosotoxicosis, can also be widely used in the scientific research to chicken.
(3) present invention first separation obtains peripheral white blood cells, then is dyed with fluorescence monoclonal antibody, because chicken red blood cell has
Core, it is necessary to obtain leucocyte with lymphocyte separation medium；So result in abundant peripheral white blood cells.
(4) in the present invention during machine testing, in the two-dimentional quadrantal diagrams of FSC/SSC, lymphocyte cumularsharolith is in close to FSC axle positions
Put；By parameter regulation, make lymphocyte and along SSC axles, monocyte and granulocyte away from FSC axles are separated；So do energy
It is enough significantly to separate lymphocyte with other cells.
(5) the CD8 positive cells of chicken are without obvious strong, weakly positive point group, the present invention can rationally distinguish CD3, CD4 and
CD8 positive cells, the percentage accounting of each subgroup T lymphocytes of reasonable analysis.
Certainly, any product for implementing the present invention it is not absolutely required to while reaching all the above technique effect.
Brief description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes the part of the present invention, this hair
Bright schematic description and description is used to explain the present invention, does not constitute inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 is medium size lymphocyte separating effect figure of the embodiment of the present invention, wherein：(A) divide to centrifuge prolymphocyte
Chaotropic and anticoagulation are demarcated clearly separating effect figure；(B) mixed to centrifuge prolymphocyte separating liquid with anticoagulation
Separating effect figure；Before a is centrifugation, after b is centrifugation；
Fig. 2 is that the embodiment of the present invention takes chicken PBLC schematic diagram in the two-dimentional scatter diagram centre circles of FSC/SSC；
Fig. 3 is oral contamination AFB in the embodiment of the present invention1Chicken T lymphocyte subsets interpretation of result figure.
Embodiment
Describe embodiments of the present invention in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section can fully understand and implement according to this to solve technical problem and reach the implementation process of technology effect.
The method of Flow cytometry chicken T lymphocyte subsets of the present invention, specifically includes following steps：
Step 1, the peripheric venous blood 2mL of tested chicken is taken, anticoagulation is prepared, is handled with lymphocyte separation medium and obtains periphery
Blood leukocytes；
Further, described handled with lymphocyte separation medium obtains peripheral white blood cells, is specially：Pressed in streaming pipe
According to volume ratio 1:1 successively adds lymphocyte separation medium and anticoagulation, it is ensured that anticoagulation and lymphocyte separation medium boundary are clear,
Avoid the two from mixing as far as possible, peripheral white blood cells are obtained after centrifugation.
As shown in Figure 1A, anticoagulation is carefully added into above lymphocyte separation medium, makes separating liquid and anticoagulation before centrifugation
Between demarcate clear, then can obtain the cotton-shaped intermediate layer cell of more amount milky (i.e. peripheral white blood cells layer) after centrifuging, and with
Upper strata boundary is clear (→ shown).If the two has mixing, liquid layered effect after centrifugation can be made not good, can be influenceed to some extent
The pick-up rate of lymphocyte；As shown in Figure 1B, when the anticoagulation of addition has obvious mix with lower floor lymphocyte separation medium, then from
The cotton-shaped intermediate layer thickness obtained after the heart is thinning, and unclear (→ shown) with supernatant liquor boundary.Therefore, in separation chicken peripheral blood
During lymphocyte, method that be as shown in Figure 1A adds blood above lymphocyte separation medium, and the two is avoided as far as possible
Mixing, it is ensured that peripheric venous blood and lymphocyte separation medium boundary are clear, and centrifuge in time, to ensure to obtain enough peripheral bloods
Leucocyte.
Wherein, the rotating speed of centrifugation is 2500r/min, and the time is 15min.Liquid is divided into three layers, middle breast after centrifugation
White flock liquid level is peripheral white blood cells layer.
Step 2, with PBS liquid to the peripheral white blood cells centrifuge washing after, prepare single cell suspension；
Further, the single cell suspension for preparing is specially：Intermediate layer cell is carefully drawn with liquid-transfering gun in another stream
In formula pipe, 4 DEG C of PBS liquid of addition 1mL precoolings, centrifuge washing after mixing, it is 1 × 10 to abandon after supernatant and adjust cell concentration6-1×
107Individual/mL single cell suspension, is saved backup in 4 DEG C.Single cell suspension excessive concentration can cause dyeing insufficient；Concentration mistake
Low, cell flow rate is low when can make machine testing, influences the accuracy of testing result.
Wherein, the rotating speed of centrifuge washing is 600-1000 r/min, time 5min.
Step 3, draw single cell suspension described in 100 μ L and in another streaming pipe, be separately added into anti-chicken CD3, CD4, CD8 mono-
Clonal antibody, vortex is mixed, in 4 DEG C of lucifuge dyeing 30min, as detection pipe；
Meanwhile, single cell suspension described in 100 μ L is drawn in another streaming pipe, is used as negative set and is managed, in case streaming is thin
Negative area is set on born of the same parents' instrument during machine testing.
Further, the negative processing mode for setting pipe has two kinds, and one is to use Isotype control reagent dyeing；Two be not contaminate
Color.
Further, the system of selection of Isotype control reagent is specially：The primary antibody of selection surface marker corresponding with antibody is complete
The antibody of exactly the same Species origin, identical hypotype and fluorescence labeling.For example, detection pipe uses the CD4 FITC of the anti-chicken of mouse
It is Mouse IgG1 that its composition is shown on the antibody of mark, specification, then Isotype control is exactly the Mouse of FITC marks
IgG1.It is familiar with after point public sentiment condition of each monoclonal antibody positive cell, negative control area is set usually using blank control.
Step 4, after dyeing, washed with 500 μ L precooling PBS liquid and cell is resuspended, detected using flow cytometer；
Further, detected in the step 4 using flow cytometer, including：In forward scattering light/lateral scattering
Voltage and current linear gain (AMP) parameter is adjusted in light (FSC/SSC) two-parameter figure, makes lymphocyte and peripheral cell point
Open, as shown in Figure 2 A；Choose lymphocyte and gating；Negative area is set；Compensated with detection pipe regulation fluorescence, complete data and obtain
Take.
In the present embodiment, in detection, cell liquid is placed in loading mouthful, flow cytometer is run with low-speed mode.
Wherein, described regulation voltage and current linear gain (AMP) parameter, be specially：Side scattered light (SSC) is adjusted to lead to
The voltage in road, makes cell be divided into obvious two along SSC direction of principal axis；The AMP parameters of forward scattering light (FSC) passage are adjusted,
Separate the cell fragment of cell mass and left side rocket-shaped.According to the rule that chicken T lymphocyte differentiations are ripe, CD3 in peripheral blood+
CD4+And CD3+CD8+T lymphocyte ratios are relatively more, and CD4+CD8+T lymphocyte ratios are less, and correct regulation is each accordingly
The fluorescence compensation of passage.
Wherein, the selection lymphocyte is specially：Gating circle takes the cell mass close to forward scattering optical axis.
Wherein, it is described that negative area is set, be specially：Guan Shangji is set with feminine gender, in CD3/CD4, CD3/CD8 and CD4/
In CD8 two-parameter figure, the cell in the regulation cross door lower left corner makes its percentage be more than 98%.
In step 4, after the compensation of regulation fluorescence, specific accessible effect is：In CD4/CD8 scatter diagrams, CD4+CD8+
T lymphocyte ratios are typically less than 3%；In CD3/CD4 and CD3/CD8 scatter diagrams, the CD4 or CD8 in the cross door lower right corner are mono-
Positive cell percentage is typically less than 2%.
Step 5, testing result is analyzed, chicken T lymphocyte subsets ratio is obtained.
Further, testing result is analyzed in the step 5, including：Negative area's setting figure is recalled, the tool of cross door is determined
Body position；Testing result figure is recalled, T lymphocytes and gating are taken in the two-parameter figure centre circles of FSC/SSC；Again respectively CD3/CD4,
Double-negative area, single negative area and double hot spots are accurately defined in CD3/CD8, CD4/CD8 quadrantal diagram, CD3 is read respectively+、CD3+
CD4+And CD3+CD8+T cent lymphocytes, so as to obtain chicken T lymphocyte subsets ratio；Calculate CD3+CD4+With
CD3+CD8+The ratio of T lymphocytes.
Specifically, correct circle takes T lymphs thin in the two-parameter figures of FSC/SSC using CellQuest or FlowJo softwares
Born of the same parents and gating.The gating position of lymphocyte is chosen during analysis can cause result difference greatly, should enclose the cell taken close to SSC axles
Group and gating, as shown in Figure 2 A.When circle takes target cell as shown in Fig. 2A, CD3 positive t lymphocytes are shown in right hand quadrant figure
Accounting example is more；When circle takes target cell as shown in Fig. 2 B, then wherein CD3 positive cells ratio is less.Therefore, because peripheral blood
Ripe CD3 positive t lymphocytes accounting example is more in lymphocyte, and method that should be as shown in Fig. 2A is in FSC/SSC scatter diagrams
Centre circle takes aim cell, i.e. circle to take the cell mass close to SSC axles.
Wherein, double-negative area, single negative area and double hot spots are accurately defined, is specially：It is big according to negative area's cell quantity
In 98% standard, the position of cross door is determined with CD3/CD4, CD3/CD8, CD4/CD8 quadrantal diagram for the cell that is unstained, thus
Cross door position of the staining cell in corresponding quadrantal diagram is determined, the cross door lower left corner shows double-negative area's cell proportion, upper left
Angle and the single positive cell ratio of lower right corner display, the upper right corner shows double positive cells ratio.
It is described to read CD3 respectively+、CD3+CD4+And CD3+CD8+T cent lymphocytes, be specially：CD3+T lymphocytes
The value of percentage is the upper left corner and upper right corner cell percentages summation in CD3/CD4 quadrantal diagrams；CD3+CD4+And CD3+CD8+T drenches
The value of bar cell percentages is respectively the cell percentages in the upper right corner in CD3/CD4 and CD3/CD8 quadrantal diagrams.
It is different from detection mammal main when with Flow cytometry chicken T lymphocyte subsets
Including：One be chicken red blood cell have core, it is necessary to use lymphocyte separation medium obtain leucocyte；Two be in the two-dimentional quadrants of FSC and SSC
In figure, correct geosphere to take lymphocyte populations to be analyzed；Three be fluorescence compensation appropriate regulation and positive cell position just
Really choose has considerable influence to experimental result.The present invention avoids blood thin with lymph by being tried one's best when separating peripheral white blood cells
Born of the same parents' separating liquid is dissolved each other, to obtain enough lymphocytes；In detection, it should correctly enclose and take lymphocyte populations, reasonable adjusting voltage
Compensated with AMP parameters, and reasonable adjusting fluorescence；It is sub- so as to be successfully established a kind of chicken periphery blood T lymphocyte of stable specification
The flow cytomery method of group.
Embodiment
The oral contamination AFB of detection1Chick fresh peripheral blood in CD3/CD4/CD8 expression.
Test chicken is equally divided into two groups, control group chick feeding normal control daily ration, AFB1Group chick feeding contains certain agent
Measure horizontal AFB1Daily ration.After feeding 21 days, every group randomly selects 6 chickens, and jugular vein blood collection prepares anticoagulation, for detecting
The T lymphocyte subsets distribution situation of chicken.
Step 1, prior to addition 1mL lymphocyte separation mediums in streaming pipe, then isometric new blood is carefully added into, made
New blood is fully located at lymphocyte separation medium upper strata, it is to avoid the two mixing；
Step 2, streaming pipe is placed in common supercentrifuge, 15min is centrifuged with 2500r/min rotating speed；
Step 3, after the completion of centrifugation, liquid is divided into three layers, carefully drawn with liquid-transfering gun the cotton-shaped liquid of intermediate layer milky in
In another streaming pipe, the PBS liquid (4 DEG C) of 1mL precoolings is added, with 600-1000r/min rotating speed centrifuge washing 5min after mixing,
Supernatant is abandoned, then is resuspended with PBS liquid, adjustment cell concentration is 1 × 106-1×107Individual/mL, obtains single cell suspension；
Step 4, the μ L of single cell suspension 100 prepared are drawn respectively in other two streaming pipes, and a branch pipe puts 4 DEG C of guarantors
Deposit standby as negative and pipe is set；Another Zhi Zuowei detection pipes, are separately added into the anti-chicken CD3-SPRD (SBA, USA) of mouse, the anti-chicken of mouse
Each 1 part of anti-chicken CD8a-PE (SBA, the USA) monoclonal antibody of CD4-FITC (SBA, USA), mouse, vortex blending instrument is mixed rapidly,
Lucifuge dyeing 30min is stood in 4 DEG C；
Step 5, after dyeing, 500 μ L precooling PBSs are added into above-mentioned two streaming pipes, streaming is used after mixing
Cell instrument (BD FACSCalibur) is detected：Flow cytometer is run with low-speed mode (LOW Speed)；In FSC/SSC
The AMP parameters of the voltage of correct regulation SSC passages and FSC passages, make cell be divided into obvious two, such as scheme in two-parameter figure
Shown in 3；Choose the cell mass and gating close to SSC axles；Pipe is set to set negative area with feminine gender；Mended with detection pipe regulation fluorescence
Repay, complete the data acquisition of sample.
Step 6, testing result is analyzed
Using FlowJo7.6 softwares T lymphocytes and gating are taken in the two-parameter figure centre circles of FSC/SSC；Again respectively in CD3/
Press the standard that negative area cell quantity is less than 98% in mark signature door in CD4, CD3/CD8 and CD4/CD8 quadrantal diagram, cross door position
Setting, and then obtain CD3+、CD3+CD4+、CD3+CD8+T lymphocyte percentage values.
Experimental result is as shown in figure 3, ingest containing AFB1CD3 in the chick peripheral blood of daily ration+And CD3+CD8+T lymphocytes
Ratio is reduced.As a result show, AFB1Its cell can be disturbed by reducing the distribution of mature T lymphocyte subgroup in chicken peripheral blood
Immunologic function.
The present invention can be seen that by trying one's best when separating peripheral white blood cells and avoiding blood and lymph by above content
Cell separation liquid dissolves each other, to obtain enough lymphocytes；In detection, it should correctly enclose and take lymphocyte populations and reasonable adjusting electricity
Pressure and fluorescence compensation；So as to be successfully established a kind of flow cytometer inspection of the chicken T lymphocyte subsets of stable specification
Survey method, it is to avoid the error that human factor is caused in process of the test.Chicken peripheral blood T is more intuitively shown using flow cytometry
Point group of lymphocyte subgroup, reliable Technical Reference is provided for the research of chicken T lymphocyte subsets.
Some vocabulary have such as been used to censure special component or method among specification and claim.Art technology
Personnel are, it is to be appreciated that different regions may call same composition with different nouns.This specification and claims are not
In the way of the difference of title is used as differentiation composition.As the "comprising" of the specification in the whole text and claim mentioned in is
One open language, therefore " include but be not limited to " should be construed to." substantially " refer in receivable error range, this area
Technical staff can solve the technical problem in the range of certain error, basically reach the technique effect.Specification is follow-up
It is described as implementing the better embodiment of the present invention, so description is for the purpose of illustrating the rule of the present invention, not
To limit the scope of the present invention.Protection scope of the present invention is worked as to be defined depending on the appended claims person of defining.
It should also be noted that, term " comprising ", "comprising" or its any other variant are intended to nonexcludability
Comprising, so that commodity or system including a series of key elements not only include those key elements, but also including without clear and definite
Other key elements listed, or also include for this commodity or the intrinsic key element of system.In the feelings of not more limitations
Under condition, the key element limited by sentence "including a ...", it is not excluded that in the commodity or system including the key element also
There is other identical element.
Some preferred embodiments of invention have shown and described in described above, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, the exclusion to other embodiment is not to be taken as, and available for various other combinations, modification
And environment, and can be carried out in invention contemplated scope described herein by the technology or knowledge of above-mentioned teaching or association area
Change., then all should be in the appended power of invention and the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention
In the protection domain that profit is required.
Claims (10)
1. the method for Flow cytometry chicken T lymphocyte subsets, it is characterised in that comprise the following steps：
Step 1, chicken peripheric venous blood is taken, anticoagulation is prepared, is handled with lymphocyte separation medium and obtains peripheral white blood cells；
Step 2, with PBS liquid to the peripheral white blood cells centrifuge washing after, prepare single cell suspension；
Step 3, the single cell suspension is drawn, anti-chicken CD3, CD4, CD8 monoclonal antibody is separately added into, vortex is mixed, in 4 DEG C
Lucifuge is dyed, and is used as detection pipe；Negative set is got out simultaneously to manage；
Step 4, after dyeing, washed with PBS liquid and cell is resuspended, detected using flow cytometer；
Step 5, testing result is analyzed, chicken T lymphocyte subsets ratio is obtained.
2. the method for Flow cytometry chicken T lymphocyte subsets as claimed in claim 1, it is characterised in that
Described handled with lymphocyte separation medium obtains peripheral white blood cells, is specially：According to volume ratio 1 in streaming pipe:1 priority adds
Plus lymphocyte separation medium and anticoagulation, it is ensured that anticoagulation and lymphocyte separation medium boundary are clear, are obtained after centrifugation outer
All blood leukocytes.
3. the method for Flow cytometry chicken T lymphocyte subsets as claimed in claim 2, it is characterised in that
The rotating speed of the centrifugation is 2500r/min, and the time is 15min.
4. the method for Flow cytometry chicken T lymphocyte subsets as claimed in claim 1, it is characterised in that
The single cell suspension for preparing is specially：It is resuspended after peripheral white blood cells are washed with the PBS liquid of precooling, is adjusted to cell concentration
1×106-1×107Individual/mL single cell suspension, is saved backup in 4 DEG C.
5. the method for Flow cytometry chicken T lymphocyte subsets as claimed in claim 1, it is characterised in that
The time of the dyeing is 30min.
6. the method for Flow cytometry chicken T lymphocyte subsets as claimed in claim 1, it is characterised in that
The use flow cytometer detected, including：Voltage and electricity are adjusted in forward scattering light/two-parameter figure of side scattered light
Cleanliness gain parameter, makes lymphocyte be separated with peripheral cell；Choose lymphocyte and gating；Pipe is set to set with feminine gender cloudy
Property area, with detection pipe regulation fluorescence compensate.
7. the method for Flow cytometry chicken T lymphocyte subsets as claimed in claim 6, it is characterised in that
The regulation voltage and current linear gain parameter, be specially：Adjust lateral scattering optical channel voltage, by lymphocyte populations with
Peripheral cell is separated；The current gain of forward scattering optical channel is adjusted, cell mass is separated with cell fragment.
8. the method for Flow cytometry chicken T lymphocyte subsets as claimed in claim 6, it is characterised in that
The negative area of setting is specially：Take the negative region that pipe flow cytometer is set, cross door lower left in scatter diagram is set
For negative area, regulation voltage makes negative area's inner cell percentage be more than 98%.
9. the method for Flow cytometry chicken T lymphocyte subsets as claimed in claim 1, it is characterised in that
The analysis testing result, including：Circle takes target cell and gating, respectively in CD3/CD4, CD3/CD8, CD4/CD8 quadrantal diagram
In accurately define double-negative area, single negative area and double hot spots, read CD3+、CD3+CD4+And CD3+CD8+T lymphocyte percentages
Than so as to obtain chicken T lymphocyte subsets ratio.
10. the method for Flow cytometry chicken T lymphocyte subsets as claimed in claim 9, it is characterised in that
The circle takes target cell and gating requires that circle takes the cell mass close to forward scattering optical axis.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107941677A (en) * | 2017-10-25 | 2018-04-20 | 谭文勇 | A kind of circulating endothelial cell number hypotype detection and separator |
| CN110488009A (en) * | 2019-04-12 | 2019-11-22 | 北京市理化分析测试中心 | A kind of detection method and application of the immune function of human body of non-disease diagnostic purpose |
| WO2020107496A1 (en) * | 2018-12-01 | 2020-06-04 | 铭道创新（北京）医疗技术有限公司 | Flow cytometry testing method for lymphocyte in immune cell |
| CN111527406A (en) * | 2018-12-01 | 2020-08-11 | 铭道创新（北京）医疗技术有限公司 | Preparation method of lymphocyte sample for flow cytometry analysis |
| CN114428050A (en) * | 2022-01-27 | 2022-05-03 | 南京大学 | Flow cytometry detection method suitable for detecting cell endogenous factors by using fluorescent probe |
-
2017
- 2017-05-09 CN CN201710322064.7A patent/CN107063982A/en active Pending
Non-Patent Citations (8)
| Title |
|---|
| 余能伟 等: "帕金森病患者外周血T淋巴细胞检测及临床意义", 《四川医学》 * |
| 吕世静 等: "《临床免疫学检验》", 31 August 2015, 中国医药科技出版社 * |
| 左连富: "《流式细胞术样品制备技术》", 31 August 1991, 华夏出版社 * |
| 杨晓璐: "重组鸡α干扰素/白细胞介素-2融合蛋白体外活性及体内最佳使用剂量研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 罗荣辉 等: "《生物医学光子学》", 31 August 2008, 吉林大学出版社 * |
| 谢昆 等: "鸡外周血淋巴细胞的分离和体外培养", 《中国家禽》 * |
| 郭焱 等: "《免疫学教程》", 31 December 2015, 清华大学出版社 * |
| 金晓 等: "壳聚糖对肉仔鸡外周血T淋巴细胞亚群的影响", 《饲料工业》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107941677A (en) * | 2017-10-25 | 2018-04-20 | 谭文勇 | A kind of circulating endothelial cell number hypotype detection and separator |
| WO2020107496A1 (en) * | 2018-12-01 | 2020-06-04 | 铭道创新（北京）医疗技术有限公司 | Flow cytometry testing method for lymphocyte in immune cell |
| CN111527406A (en) * | 2018-12-01 | 2020-08-11 | 铭道创新（北京）医疗技术有限公司 | Preparation method of lymphocyte sample for flow cytometry analysis |
| CN111527395A (en) * | 2018-12-01 | 2020-08-11 | 铭道创新（北京）医疗技术有限公司 | Flow cytometry detection method for lymphocytes in immune cells |
| EP3889603A4 (en) * | 2018-12-01 | 2022-08-10 | Mingdao Innovation (Beijing) Medical-Tech Co., Ltd. | Preparation method for lymphocyte sample for flow cytometry analysis |
| CN111527395B (en) * | 2018-12-01 | 2024-01-26 | 铭道创新（北京）医疗技术有限公司 | Flow cytometry detection method for lymphocytes in immune cells |
| CN110488009A (en) * | 2019-04-12 | 2019-11-22 | 北京市理化分析测试中心 | A kind of detection method and application of the immune function of human body of non-disease diagnostic purpose |
| CN110488009B (en) * | 2019-04-12 | 2023-09-05 | 北京市理化分析测试中心 | Detection method and application of human immune function for non-disease diagnosis purpose |
| CN114428050A (en) * | 2022-01-27 | 2022-05-03 | 南京大学 | Flow cytometry detection method suitable for detecting cell endogenous factors by using fluorescent probe |
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