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Optimized cynomolgus mononuclear cell separation method

A separation method and nuclear cell technology, applied in the field of optimized cynomolgus monkey mononuclear cell separation, can solve the problems of expensive separation kits and unsatisfactory separation effect, and achieve the effect of reducing experimental cost and high cell activity.

Pending Publication Date: 2021-08-10
上海赛笠生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Monkey-derived PBMC is an important raw material in many drug testing work, but the white blood cell density of monkeys is not completely consistent with that of human, so the separation effect of directly using 1.077g / ml ficoll is not very satisfactory, and the existing Separation kits are more expensive

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] An optimized cynomolgus monkey mononuclear cell isolation method specifically comprises the following steps:

[0019] Step 1. After obtaining monkey blood, add hydroxyethyl starch, mix and settle for 20 minutes;

[0020] Step 2, dilute the 1.077g / ml ficoll solution with DPBS solution;

[0021] Step 3. Take a 50ml centrifuge tube and add 20ml of diluted ficoll solution;

[0022] Step 4. Slowly add 20ml of settled monkey blood to the ficoll solution, and keep the interface clear during the addition process;

[0023] Step 5. Centrifuge at 600g for 30 minutes at a temperature of 20°C, set the speed up to 5 minutes, and set the speed down to 10 minutes;

[0024] Step 6. After standing still, discard the upper serum layer, collect the third layer of white blood cell layer, and transfer it to another centrifuge tube;

[0025] Step 7, add DPBS solution to 50ml, centrifuge at 450g for 10min to wash away impurities;

[0026] Step 8. Resuspend the cell pellet and count.

Embodiment 2

[0028] An optimized cynomolgus monkey mononuclear cell isolation method specifically comprises the following steps:

[0029] Step 1. After obtaining monkey blood, add 1 / 4 volume of 6% hydroxyethyl starch, mix and settle for 20 minutes;

[0030] Step 2, the ficoll solution of 1.077g / ml is diluted with DPBS solution, and the volume ratio of ficoll solution and DPBS solution is 4:1;

[0031] Step 3. Take a 50ml centrifuge tube and add 20ml of diluted ficoll solution;

[0032] Step 4. Slowly add 20ml of settled monkey blood to the ficoll solution, and keep the interface clear during the addition process;

[0033] Step 5. Centrifuge at 600g for 30 minutes at a temperature of 20°C, set the speed up to 5 minutes, and set the speed down to 10 minutes;

[0034] Step 6. After standing still, discard the upper serum layer, collect the third layer of white blood cell layer, and transfer it to another centrifuge tube;

[0035] Step 7, add DPBS solution to 50ml, centrifuge at 450g for 10...

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Abstract

The invention provides an optimized cynomolgus mononuclear cell separation method, which specifically comprises the following steps: obtaining monkey blood, adding hydroxyethyl starch, uniformly mixing and settling the blood for 20 minutes; diluting the ficoll solution with a DPBS solution; preparing a 50 ml centrifugal tube, and adding 20 ml of the diluted ficoll solution; slowly adding 20ml of settled monkey blood into the ficoll solution, and paying attention to keep the interface clear; centrifuging the blood at 20 DEG C under 600 g for 30 min, and performing standing treatment; discarding an upper serum layer, collecting a third leukocyte layer, and transferring the third leukocyte layer into another centrifugal tube; adding a DPBS solution to 50 ml, and performing 450 g centrifugation for 10 min to wash away the impurities; and re-suspending the cell precipitate, and counting the cells. In the invention, the simple technical system for separating the PBMC from the peripheral blood of the cynomolgus monkey is established, wherein the experiment cost can be reduced.

Description

technical field [0001] The invention belongs to the technical field of nuclear cell separation, and in particular relates to an optimized method for separating mononuclear cells from cynomolgus monkeys. Background technique [0002] 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 work, but the white blood cell density of monkeys is not completely consistent with that of human, so the separation effect of directly using 1.077g / ml ficoll is not very satisfactory, and the existing Separation kits are more expensive. Contents of the invention [0003] The purpose of the present invention is to provide an optimized method for isolating mononuclear cells from cynomolgus monkeys, so as to solve the problems raised in the above-mentioned background technology. [0004] In order to achieve the above object, the present invention...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2509/00
Inventor 张英博
Owner 上海赛笠生物科技有限公司
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