CN113049815A - Flow type gate-circling method for cynomolgus monkey lymphocytes - Google Patents
Flow type gate-circling method for cynomolgus monkey lymphocytes Download PDFInfo
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- 241000282567 Macaca fascicularis Species 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 30
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 36
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims abstract description 23
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims abstract description 21
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 claims description 37
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims description 31
- 210000004369 blood Anatomy 0.000 claims description 24
- 239000008280 blood Substances 0.000 claims description 24
- 210000004027 cell Anatomy 0.000 claims description 13
- 238000011534 incubation Methods 0.000 claims description 10
- 239000003219 hemolytic agent Substances 0.000 claims description 9
- 210000000601 blood cell Anatomy 0.000 claims description 4
- 210000003743 erythrocyte Anatomy 0.000 claims description 4
- 238000001514 detection method Methods 0.000 abstract description 8
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 11
- 210000000265 leukocyte Anatomy 0.000 description 8
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- 210000000440 neutrophil Anatomy 0.000 description 7
- 210000003651 basophil Anatomy 0.000 description 4
- 210000003979 eosinophil Anatomy 0.000 description 4
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- 238000011161 development Methods 0.000 description 2
- 230000008054 signal transmission Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000004366 CD4-positive T-lymphocyte Anatomy 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
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Abstract
The invention discloses a flow type gate enclosing method for cynomolgus monkey lymphocytes. The flow-type gate-circling method utilizes CD45 to circle out all lymphocytes, so that all lymphocytes are circled out to the maximum extent, and the purity of the circled lymphocytes is ensured; and the CD4 is circled by using the CD4 and the CD8+And CD8+T lymphocytes, trapping as much as possible all CD4+T lymphocytes and CD8+T lymphocytes and ensures the trapped CD4+T lymphocytes and CD8+Purity of T lymphocytes; the flow type gate circling method of the invention does not depend on the experience of technical personnel excessively, ensures the consistency of detection and ensures that the gate circling method is more reasonable and objective.
Description
Technical Field
The invention belongs to the field of cell flow detection, and particularly relates to a flow type circle gate method of cynomolgus monkey lymphocytes.
Background
The flow cytometry detection of the cynomolgus monkey has the main function of evaluating the change of the lymphocyte function of the cynomolgus monkey in the preclinical safety evaluation of medicaments. The current conventional T lymphocyte flow gating method is to first cycle all lymphocytes through the experience of the skilled person (fig. 1), and then perform the subsequent operation through the CD3/CD4/CD8 antibody, and the experience of the skilled person determines the quality of the lymphocyte gating.
The CD45 molecule is expressed on all leukocytes, is called Leukocyte Common Antigen (LCA), CD45 is composed of a class of transmembrane proteins with similar structures and larger molecular weights, is widely present on the surface of leukocytes, has the function of protein tyrosine phosphatase in a cytoplasmic segment, can ensure that tyrosine on substrates P56lck and P59fyn is dephosphorylated and activated, plays an important role in cell information transmission, and has important significance in development and maturation, function regulation and signal transmission of lymphocytes because CD45 is a key molecule of signal transmission on cell membranes.
The most common T lymphocyte circle approaches are currently: the cells are classified into neutrophils, monocytes and lymphocytes by using forward light FSC and side light SSC of a flow cytometer. The technician empirically circles the lymphocytes, and then performs the subsequent operation by fluorescence of antibodies such as CD3/CD4/CD 8. The drawback of this circle of door mode is that the cells circled out in the door are lymphocytes, and because of the different experience of the detection personnel, the purity of the lymphocytes in the door is different. Especially when the abundance of lymphocytes is low, the loss of lymphocytes is observed, and the result is influenced.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of low yield and low accuracy caused by subjectivity in lymphocyte gate enclosing in the prior art, and provide a flow-type gate enclosing method for cynomolgus monkey lymphocytes, wherein all leukocytes are enclosed by utilizing CD45 molecules, and T lymphocytes are enclosed by CD4 and CD8, so that the T lymphocytes are ensured to be identified to the maximum extent, the condition that the conventional gate enclosing method depends too much on the experience of detection personnel is changed, the detection is more standardized, and the quality of the test is ensured.
In order to solve the problems, the invention adopts the technical scheme that: a flow-type circumcision method of cynomolgus monkey lymphocytes comprises the following steps: cynomolgus monkey whole blood cells incubated with the CD45 antibody were detected by lateral light and CD45 fluorescence, trapping lymphocytes in cynomolgus monkey whole blood.
Preferably, the flow gating method further comprises the step of using CD4 fluorescence to coil CD4 in the cynomolgus monkey whole blood incubated by the CD4 antibody+T lymphocytes.
Preferably, the flow gating method further comprises the step of using CD8 fluorescence to coil CD8 in the cynomolgus monkey whole blood after incubation by the CD8 antibody+T lymphocytes.
Preferably, the incubation time is 45 min.
Preferably, the flow gate method further comprises removing red blood cells from the cynomolgus monkey whole blood after the incubating. Among them, the method for removing erythrocytes from cynomolgus monkey whole blood is preferably incubation with a hemolytic agent.
Preferably, the incubation with the hemolytic agent is for a period of 20 min.
Preferably, the flow gate method comprises the following steps:
1) adding an anti-monkey CD45 antibody, a CD4 antibody and a CD8 antibody into cynomolgus monkey whole blood, and incubating in a dark place;
2) adding hemolytic agent into the cynomolgus monkey whole blood treated in the step 1);
3) adjusting the voltage of each fluorescence channel of the flow cytometer to obviously classify the cells into 5 types, and setting the voltage as a template; detecting the cynomolgus monkey whole blood treated in the step 2) by using lateral light of a flow cytometer and CD45 fluorescence, and trapping lymphocytes;
4) fluorescence of CD4 and CD8 was used to coil CD4+T lymphocytes and CD8+T lymphocytes.
In step 3), the voltage setting parameters are as follows:
flow cytometer acquisition parameter settings-gain item settings
Preferably, the incubation time in step 1) is 45 min.
According to the invention, the CD45 antibody, the CD4 antibody and the CD8 antibody are incubated with whole cynomolgus monkey blood for 45min in a dark place, and after a hemolytic agent is added, incubation is carried out in a dark place for 20min, and then the detection is carried out on a computer. The volume ratio of the CD45 antibody, the CD4 antibody, or the CD8 antibody to the cynomolgus monkey whole blood may be conventional in the art, preferably 1: 10.
The cells are divided into five types by using side light SSC and CD45 fluorescence, namely neutrophils, eosinophils, basophils, monocytes and lymphocytes, and CD4 positive T lymphocytes and CD8 positive T lymphocytes are distinguished by CD4 and CD8 antibodies, so that the purity of the T lymphocytes is ensured to the maximum extent.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention. The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: all lymphocytes are circled by using CD45, so that all lymphocytes are circled to the maximum extent, and the purity of the circled lymphocytes is ensured; and the CD4 is circled by using the CD4 and the CD8+And CD8+T lymphocytes, trapping as much as possible all CD4+T lymphocytes and CD8+T lymphocytes and ensures the trapped CD4+T lymphocytes and CD8+Purity of T lymphocytes; the door closing mode does not excessively depend on the experience of technicians, the consistency of detection is ensured, and the door closing mode is more reasonable and objective.
Drawings
FIG. 1 shows the division of cynomolgus monkey whole blood cells into neutrophils, monocytes and lymphocytes using SSC and CD 45.
FIG. 2 is a scheme for the re-fractionation of CD4 using CD4 and CD8 antibodies+T lymphocytes (H1-UL) and CD8+T lymphocytes (H1-LR).
FIG. 3 is a ratio of lymphocytes to leukocytes; CD4+CD8-T lymphocytes and CD4-CD8+T showerThe proportion of lymphocytes is accounted for by the lymphocytes, respectively.
FIG. 4 shows the division of cynomolgus monkey whole blood cells into neutrophils, eosinophils, basophils, monocytes and lymphocytes using FSC and SSC; the lymphocyte P2 is circled in the lower right corner.
FIG. 5 is CD3 encircled with CD3+T lymphocytes.
FIGS. 6A and 6B are views on CD3+CD3 was reisolated by CD4 and CD8 antibodies based on T lymphocytes+CD4+T lymphocytes (H1-UL) and CD3+CD8+T lymphocytes (H1-LR) and the corresponding proportion.
FIG. 7 shows the result of hematological examination of the blood of the same cynomolgus monkey.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1
1. Antibody-treated cynomolgus whole blood
Adding 10 μ l of anti-monkey CD45 antibody, CD4 antibody and CD8 antibody into a flow tube, adding 100 μ l of cynomolgus monkey whole blood (Guangxi Guidong primate development experiment Co., Ltd.), mixing, and incubating for 45min in dark;
table 1: antibody information
After 2ml of a hemolytic agent (BD Co., U.S.A.: 9127745) was added, the cells were incubated for 20min with exclusion of light.
2. Cynomolgus monkey whole blood flow cytometer ring door
After the apparatus was set up, the cells were examined by flow cytometry (Beckman FLEX S flow cytometer, Beckman Coulter, USA). Adjusting the voltage of each fluorescence channel of the flow cytometer to obviously classify the cells into 5 types, and setting the voltage as a template (the parameters are shown in table 2); cells were classified into five classes, neutrophils, eosinophils, basophils, monocytes and lymphocytes (fig. 1), using side light SSC and CD45 fluorescence, with lymphocytes being circled.
Table 2 flow cytometer acquisition parameter settings-gain item settings
| FSC | 197 |
| SSC | 77 |
| FITC | 134 |
| PE | 568 |
| PC5.5 | 577 |
| APC | 110 |
CD4 was further distinguished from the trapped lymphocytes by CD4 and CD8 antibodies+T lymphocytes and CD8+T lymphocytes (FIG. 2), and CD4 was obtained+CD8-/CD4-CD8+The ratio of (a) to (b).
The results showed that lymphocytes accounted for 50 of the leukocytes78%, wherein CD4+CD8-T lymphocytes account for 33.73%, CD4-CD8+T lymphocytes account for 34.90% (fig. 3).
The same vessel was tested hematologically and lymphocytes accounted for 60.5% of the leukocytes, similar to the results with the CD45 circle gate (FIG. 7).
The hematology test is carried out by using Siemens ADVIA2120 instrument (relevant parameters are set according to the specification of the measuring instrument), and the instrument classifies the white blood cells into neutrophils, lymphocytes, monocytes, eosinophils and basophils by adopting myeloperoxidase staining technology and flow cytometry.
Comparative example 1
1. Antibody-treated cynomolgus whole blood
Mu.l of each of the anti-monkey CD3 antibody, the CD4 antibody and the CD8 antibody was added to the flow tube, 100. mu.l of cynomolgus monkey whole blood was added thereto, and the mixture was mixed and incubated for 45min in the dark.
After adding 2ml of hemolytic agent, incubation was performed for 20min in the dark.
2. Cynomolgus monkey whole blood flow cytometer ring door
After the apparatus was set up, the white blood cells were separated into neutrophils, monocytes and lymphocytes by FSC and SSC, and the lymphocytes P1 (43.48% of white blood cells) were circled according to the experience of the examiner (FIG. 4).
Cell subdivision into CD3 in the P1 Gate with CD3 antibody+And CD3-The cell, wherein P2 is CD3+T lymphocytes (fig. 5).
CD4 is distinguished by CD4 and CD8 antibodies+CD8-T lymphocytes (H1-UL) and CD4-CD8+T lymphocytes (H1-LR); wherein CD4+CD8-53.7% of T lymphocytes, CD4-CD8+T lymphocytes were 40.95% (fig. 6A, 6B).
Claims (10)
1. A flow gating method of cynomolgus monkey lymphocytes is characterized by comprising the following steps: cynomolgus monkey whole blood cells incubated with the CD45 antibody were detected by lateral light and CD45 fluorescence, trapping lymphocytes in cynomolgus monkey whole blood.
2. The flow gating method of cynomolgus monkey lymphocytes of claim 1, wherein said flow gating method further comprises trapping CD4 in cynomolgus monkey whole blood incubated with CD4 antibody with CD4 fluorescence+T lymphocytes.
3. The flow gating method of cynomolgus monkey lymphocytes of claim 1 or 2, wherein the flow gating method further comprises trapping CD8 in cynomolgus monkey whole blood incubated with CD8 antibody with CD8 fluorescence+T lymphocytes.
4. The method of claim 1 to 3, wherein the incubation is for 45 min.
5. The method of any one of claims 1 to 4, further comprising removing red blood cells from cynomolgus monkey whole blood after said incubating.
6. The method of claim 5, wherein the method for removing red blood cells from cynomolgus monkey whole blood comprises incubating with a hemolytic agent.
7. The method of claim 6, wherein the incubation with the hemolytic agent is performed for a period of 20 min.
8. The flow gating method of cynomolgus monkey lymphocytes of any one of claims 1 to 7, wherein the flow gating method comprises:
1) adding an anti-monkey CD45 antibody, a CD4 antibody and a CD8 antibody into cynomolgus monkey whole blood, and incubating in a dark place;
2) adding hemolytic agent into the cynomolgus monkey whole blood treated in the step 1);
3) adjusting the voltage of each fluorescence channel of the flow cytometer to classify the cells into 5 types, and setting the voltage as a template; detecting the cynomolgus monkey whole blood treated in the step 2) by using lateral light of a flow cytometer and CD45 fluorescence, and trapping lymphocytes;
4) CD4 was fluorescence-trapped using CD4 and CD8+T lymphocytes and CD8+T lymphocytes.
9. The method of claim 8, wherein the volume ratio of the CD45 antibody, the CD4 antibody, or the CD8 antibody to the cynomolgus monkey whole blood is 1: 10.
10. The flow-through circle gate method of claim 8 or 9, wherein the incubation in step 1) with light is carried out for 45 min.
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| WO2023046111A1 (en) * | 2021-09-23 | 2023-03-30 | 上海市第一人民医院 | Basophil activation detection method and application thereof |
| CN117191515A (en) * | 2023-09-08 | 2023-12-08 | 四川大学华西医院 | Method for detecting lung cancer tumor infiltrating lymphocyte subpopulation |
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Cited By (3)
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| WO2023046111A1 (en) * | 2021-09-23 | 2023-03-30 | 上海市第一人民医院 | Basophil activation detection method and application thereof |
| CN114441419A (en) * | 2022-01-29 | 2022-05-06 | 杭州翔宇医学检验实验室有限公司 | Stream type gate circling method and application |
| CN117191515A (en) * | 2023-09-08 | 2023-12-08 | 四川大学华西医院 | Method for detecting lung cancer tumor infiltrating lymphocyte subpopulation |
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