CN113234673A - Optimized separation method for cynomolgus monkey mononuclear cells - Google Patents
Optimized separation method for cynomolgus monkey mononuclear cells Download PDFInfo
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- CN113234673A CN113234673A CN202110527154.6A CN202110527154A CN113234673A CN 113234673 A CN113234673 A CN 113234673A CN 202110527154 A CN202110527154 A CN 202110527154A CN 113234673 A CN113234673 A CN 113234673A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention provides an optimized separation method of cynomolgus monkey mononuclear cells, which specifically comprises the following steps: after obtaining monkey blood, adding hydroxyethyl starch, uniformly mixing and settling for 20 min; diluting the ficoll solution by using a DPBS solution; taking a 50ml centrifuge tube, and adding 20ml of diluted ficoll solution; slowly add 20ml of settled monkey blood to ficoll solution and take care to keep the interface clear; centrifuging at 600g for 30min at 20 ℃; after standing, removing the upper serum layer, collecting the leukocyte layer on the third layer, and transferring to another centrifuge tube; adding DPBS solution to 50ml, centrifuging for 10min at 450g, and washing off impurities; resuspend the cell pellet and count. The invention establishes a simpler technical system for separating PBMC from peripheral blood of the cynomolgus monkey, and can reduce the experiment cost.
Description
Technical Field
The invention belongs to the technical field of nuclear cell separation, and particularly relates to an optimized separation method of cynomolgus monkey mononuclear cells.
Background
Mononuclear Cells (PBMCs) are white blood cells with a single nucleus in the peripheral blood, including lymphocytes and monocytes. Monkey-derived PBMC is an important raw material in many drug testing works, but the leukocyte density of monkeys is not completely consistent with that of human-derived leukocytes, so that the separation effect of 1.077g/ml ficoll directly used is not ideal, and the existing separation kit is expensive.
Disclosure of Invention
The invention aims to provide an optimized separation method of cynomolgus monkey mononuclear cells, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: an optimized separation method of cynomolgus monkey mononuclear cells specifically comprises the following steps:
step 1, adding hydroxyethyl starch after obtaining monkey blood, uniformly mixing and settling for 20 min;
step 2, diluting 1.077g/ml of ficoll solution by using a DPBS solution;
step 3, taking a 50ml centrifuge tube, and adding 20ml of diluted ficoll solution;
step 4, slowly adding 20ml of settled monkey blood into the ficoll solution, and taking care to keep the interface clear in the adding process;
step 5, centrifuging at the temperature of 20 ℃ for 30min at 600g, setting the speed to be increased to 5min, and setting the speed to be decreased to 10 min;
step 6, after standing, removing the upper serum layer, collecting the leukocyte layer on the third layer, and transferring the leukocyte layer into another centrifuge tube;
step 7, adding the DPBS solution to 50ml, centrifuging for 10min at 450g, and washing out impurities;
step 8, resuspend the cell pellet and count.
Preferably, the hydroxyethyl starch in step 1 is 1/4 vol% 6% hydroxyethyl starch.
Preferably, the volume ratio of the ficoll solution to the DPBS solution in the step 2 is 4: 1.
Preferably, the steps 1-8 are performed in a class II biosafety cabinet, and strict adherence to aseptic procedures is achieved.
The invention has the technical effects and advantages that: the invention establishes a simpler technical system for separating PBMC from peripheral blood of the cynomolgus monkey, can obtain PBMC with high cell activity and reduce the experiment cost.
Detailed Description
Example 1
An optimized separation method of cynomolgus monkey mononuclear cells specifically comprises the following steps:
step 1, adding hydroxyethyl starch after obtaining monkey blood, uniformly mixing and settling for 20 min;
step 2, diluting 1.077g/ml of ficoll solution by using a DPBS solution;
step 3, taking a 50ml centrifuge tube, and adding 20ml of diluted ficoll solution;
step 4, slowly adding 20ml of settled monkey blood into the ficoll solution, and taking care to keep the interface clear in the adding process;
step 5, centrifuging at the temperature of 20 ℃ for 30min at 600g, setting the speed to be increased to 5min, and setting the speed to be decreased to 10 min;
step 6, after standing, removing the upper serum layer, collecting the leukocyte layer on the third layer, and transferring the leukocyte layer into another centrifuge tube;
step 7, adding the DPBS solution to 50ml, centrifuging for 10min at 450g, and washing out impurities;
step 8, resuspend the cell pellet and count.
Example 2
An optimized separation method of cynomolgus monkey mononuclear cells specifically comprises the following steps:
step 1, after obtaining monkey blood, adding 1/4 volume percent of 6 percent hydroxyethyl starch, uniformly mixing and settling for 20 min;
step 2, diluting 1.077g/ml of ficoll solution by using a DPBS solution, wherein the volume ratio of the ficoll solution to the DPBS solution is 4: 1;
step 3, taking a 50ml centrifuge tube, and adding 20ml of diluted ficoll solution;
step 4, slowly adding 20ml of settled monkey blood into the ficoll solution, and taking care to keep the interface clear in the adding process;
step 5, centrifuging at the temperature of 20 ℃ for 30min at 600g, setting the speed to be increased to 5min, and setting the speed to be decreased to 10 min;
step 6, after standing, removing the upper serum layer, collecting the leukocyte layer on the third layer, and transferring the leukocyte layer into another centrifuge tube;
step 7, adding the DPBS solution to 50ml, centrifuging for 10min at 450g, and washing out impurities;
step 8, resuspend the cell pellet and count.
Preferably, the steps 1-8 are performed in a class II biosafety cabinet, and strict adherence to aseptic procedures is achieved.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Claims (4)
1. An optimized separation method of cynomolgus monkey mononuclear cells is characterized in that: the method specifically comprises the following steps of,
step 1, adding hydroxyethyl starch after obtaining monkey blood, uniformly mixing and settling for 20 min;
step 2, diluting 1.077g/ml of ficoll solution by using a DPBS solution;
step 3, taking a 50ml centrifuge tube, and adding 20ml of diluted ficoll solution;
step 4, slowly adding 20ml of settled monkey blood into the ficoll solution, and taking care to keep the interface clear in the adding process;
step 5, centrifuging at the temperature of 20 ℃ for 30min at 600g, setting the speed to be increased to 5min, and setting the speed to be decreased to 10 min;
step 6, after standing, removing the upper serum layer, collecting the leukocyte layer on the third layer, and transferring the leukocyte layer into another centrifuge tube;
step 7, adding the DPBS solution to 50ml, centrifuging for 10min at 450g, and washing out impurities;
step 8, resuspend the cell pellet and count.
2. The method of claim 1, wherein the isolation of cynomolgus monkey mononuclear cells comprises: the hydroxyethyl starch in step 1 was 1/4 vol% 6% hydroxyethyl starch.
3. The method of claim 1, wherein the isolation of cynomolgus monkey mononuclear cells comprises: the volume ratio of the ficoll solution to the DPBS solution in the step 2 is 4: 1.
4. The method of claim 1, wherein the isolation of cynomolgus monkey mononuclear cells comprises: the steps 1-8 must be performed in a class II biosafety cabinet, and the aseptic operation is strictly followed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113249323A (en) * | 2021-05-14 | 2021-08-13 | 上海赛笠生物科技有限公司 | Novel granulocyte separation method |
| CN113755435A (en) * | 2021-10-15 | 2021-12-07 | 苏州药明康德新药开发有限公司 | Macaca fascicularis PBMC separation method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113249323A (en) * | 2021-05-14 | 2021-08-13 | 上海赛笠生物科技有限公司 | Novel granulocyte separation method |
| CN113755435A (en) * | 2021-10-15 | 2021-12-07 | 苏州药明康德新药开发有限公司 | Macaca fascicularis PBMC separation method |
| CN113755435B (en) * | 2021-10-15 | 2023-10-13 | 苏州药明康德新药开发有限公司 | Separation method of cynomolgus monkey PBMC |
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