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Simple PBMC separation method for flow cytometry detection

A separation method and flow cytometry technology, applied in the field of simple PBMC separation that can be used for flow cytometry detection, can solve other problems such as cell death, affecting flow analysis/sorting, red blood cell lysis, etc., to simplify operation steps and save money. Blood sample processing time, the effect of reducing instrument dependence

Inactive Publication Date: 2020-11-06
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When collecting samples in primary hospitals, there are often no experimental conditions for such operations
And if not separated, red blood cells will quickly lyse and cause the death of other cells
[0006] Although the hydroxyl starch precipitation method (HES method) is also used to separate cells, the cells separated by this method are mixed with a certain amount of red blood cells, and the hydroxyl starch adheres to the cells, which will affect the subsequent flow analysis / sorting. Cells were not isolated by hydroxystarch precipitation for flow cytometry analysis

Method used

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  • Simple PBMC separation method for flow cytometry detection
  • Simple PBMC separation method for flow cytometry detection
  • Simple PBMC separation method for flow cytometry detection

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1, the method for separating PBMC of the present invention

[0032] Take whole blood (six samples) added with anticoagulant (EDTA, its mass volume ratio to whole blood is 1.8mg / mL), and 6% hydroxyethyl starch solution (hydroxyethyl starch molecular weight: 480,000) by volume ratio Mix 5:1, let stand at 25°C for 30min, absorb the supernatant, and store at 4°C for 1-2 days to obtain PBMC suspension, which will be used for flow cytometry within 1-2 days.

Embodiment 2

[0033] Embodiment 2, the method for separating PBMC of the present invention

[0034] Whole blood (six samples) added with anticoagulant (EDTA, its mass volume ratio to whole blood is 1.8 mg / mL) was mixed with 6% hydroxyethyl starch solution (hydroxyethyl starch molecular weight: 480,000) by volume Mix at a ratio of 5:1, let stand at room temperature for 30 minutes, absorb the supernatant, and centrifuge at a speed of 200g and a temperature of 4°C for 5 minutes, remove the lower layer of cells, and resuspend 1x10 cells per 1ml 6 Add the amount of cells to the freezing solution (freezing solution consisting of 90% fetal bovine serum (FBS) + 10% dimethyl sulfoxide (DMSO)), and freeze at -150°C for 180 to 360 days to obtain frozen PBMC , within 180 to 360 days for flow cytometry.

[0035] The beneficial effects of the present invention are illustrated below through test examples.

experiment example 1

[0037] The same blood sample was divided into 3 parts respectively, and the Ficoll method, the separation method of the present invention for short-term storage (hydroxyethyl starch sedimentation method, HBS), the method of combining the separation method of the present invention for short-term storage and the Ficoll method to separate PBMC, each Six blood samples were separated by this method, and the obtained PBMC survival rate, CD4+T cell and CD8+T cell ratio were analyzed and detected.

[0038] 1. Separation method

[0039] 1) Ficoll centrifugation

[0040] Take whole blood added with anticoagulant (EDTA, whose mass volume ratio to whole blood is 1.8 mg / mL), mix with an equal volume of RPMI1640 basal medium, and add it to a centrifuge tube containing Ficoll solution (diluted blood sample and Ficoll solution The volume ratio is 2:1), after centrifugation at 800g at room temperature for 15min (the speed of the centrifuge is the lowest), the buffy coat cells were taken, wash...

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Abstract

The invention discloses a simple PBMC (peripheral blood mononuclear cell) separation method for flow cytometry detection. The method comprises the following steps: 1), taking whole blood, adding a separation solution, performing mixing, and performing standing; 2), taking the supernatant obtained in the step 1), and storing the supernatant at a temperature of 2-8 DEG C for 1-2 days to obtain a PBMC suspension; or taking the supernatant obtained in the step 1), performing centrifuging, taking lower cells, adding a preservation solution for resuspending, and performing cryopreserving for 180-360days at a temperature of -150 DEG C to obtain the cryopreserved PBMC. Compared with a common method, the method for separating PBMC has the advantages that the blood sample treatment time is greatlysaved, the operation steps are simplified, the dependence on instruments such as a centrifugal machine is reduced, and the method can be used for timely treating blood samples in wards, clinics and accident places, can be used for detecting CD4 and CD8 by flow cytometry, and has practical popularization and application values.

Description

technical field [0001] The invention specifically relates to a simple method for separating PBMCs that can be used for flow cytometry detection. Background technique [0002] PBMC (Peripheral blood mononuclear cell) refers to cells with a single nucleus in peripheral blood, including lymphocytes and monocytes. Human immune cells, such as CIK cells, DC cells, NK cells, and DC-T cells, are all induced and differentiated from PBMCs. To obtain the above immune cells, PBMCs need to be isolated from peripheral blood. [0003] Peripheral blood contains platelets, PBMCs, red blood cells, and multinucleated white blood cells. The density of PBMCs is different from that of other cells. The density of red blood cells and multinucleated white blood cells is relatively large, at 1.090kg / m 3 around, while the density of lymphocytes and monocytes is 1.075~1.090kg / m 3 , platelet is 1.030~1.035kg / m 3 . For this purpose, a kind of density between 1.075 ~ 1.092kg / m 3 Different kinds of bl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N1/38G01N1/42G01N15/14C12N5/078
CPCC12N5/0634C12N2509/00C12N2509/10G01N1/28G01N1/38G01N1/42G01N15/14G01N2015/1488
Inventor 牟大超吴沙沙周轶
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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