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Method for isolating monocytes in bovine peripheral blood

A technique for peripheral blood mononuclear cells and nucleated cells is applied in the field of cell separation, which can solve the problems of not separating bovine peripheral blood mononuclear cells, difficult to activate mononuclear cells, and no use of literature, and achieves simple and efficient separation, strong operability, time saving effect

Inactive Publication Date: 2012-01-25
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Reverse panning centrifugation can handle a large amount of blood, with high purity and high viability, and it is not easy to activate monocytes, but it requires expensive equipment and professional technicians
Density separation methods include the use of continuous density gradients and discontinuous density gradients, in which pumps and ultracentrifuges for making continuous density gradients are relatively expensive
[0003] In 1999, Jindrich Soltys and others used the method of immunomagnetic beads to separate bovine neutrophils. The cell yield was higher, the purity was more pure, and it showed better activity in many detection indicators. Magnetic bead method for isolation of bovine peripheral blood mononuclear cells has not been reported in the literature
Mononuclear cell isolation techniques using antibodies There is also flow cytometry, but there are no reports in the literature for the isolation of bovine peripheral blood mononuclear cells
The reverse centrifugal elutriation method requires a special reverse centrifugal elutriation equipment for the isolation of human peripheral blood mononuclear cells, but there is no literature using this technology to separate bovine peripheral blood mononuclear cells

Method used

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  • Method for isolating monocytes in bovine peripheral blood
  • Method for isolating monocytes in bovine peripheral blood
  • Method for isolating monocytes in bovine peripheral blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Separation of mononuclear cells in bovine peripheral blood samples

[0023] 1. Materials and reagents

[0024] 1) Anticoagulant ACD-A, the preparation method is: add 0.73g of anhydrous citric acid, 2.20g of sodium citrate dihydrate and 2.45g of dextrose monohydrate into every 100ml of water. Adjust the pH to 7.0-7.3, and filter to sterilize with a 0.22 μm filter. 1 volume of ACD-A anticoagulates 5-10 volumes of blood.

[0025] 2) OptiPrep TM Density gradient separation medium (Axis-Shield PoC, Norway).

[0026] 3) Tricine Buffer (Amresco).

[0027] 4) RPMI1640 medium (Gibco, invitrogen).

[0028] 5) Fetal bovine serum (Gibco, invitrogen).

[0029] 6) PBS (NaCl 8.00g, KCl 0.20g, NaCl 2 HPO 4 12H 2 O 2.91g, KH 2 PO 4 0.24g, add 800ml deionized water, adjust the pH to 7.4, dilute to 1000ml, and autoclave).

[0030] 2. Method

[0031] 1) Aseptically collect bovine peripheral blood, use ACD-A anticoagulant (50ml centrifuge tube, add about 7ml ACD-A, co...

Embodiment 2

[0041] The influence of embodiment 2 anticoagulants on isolated bovine peripheral blood mononuclear cells

[0042] The present invention improves the detailed steps and conditions on the basis of conventional separation methods. The first is the choice of anticoagulant, preferably ACD-A, followed by heparin, EDTA is not recommended. Then, washing of the cells must sufficiently remove platelets, residual serum, and the like. Finally, a set of model schemes were formed, including centrifugation with 50ml centrifuge tubes, washing with 15ml centrifuge tubes, and 25cm 2 The protocol of culture flask adherence has good repeatability and operability, does not require cell counting, saves experimental time and improves separation effect.

[0043] The present invention has explored the use condition of anticoagulant, comprises heparin (2mg / 10ml blood), EDTA dipotassium (K 2 EDTA, 18mg / 10ml blood) and ACD-A. It was found that there were significant differences in the number of PBMC...

Embodiment 3

[0052] Example 3 Effect of Cell Cleaning on Isolating Bovine Peripheral Blood Mononuclear Cells

[0053] In addition to exploring the selection of the anticoagulant, the present invention also explores the processing method of the isolated cells. The most critical step is to fully wash the cells. In previous experiments, it was often the case that the dissociation step was completely normal until the microscopic examination of cells dispersed among each other before adding the culture medium. However, when the complete medium was added to adhere to the wall, the cells quickly coagulated with each other, and finally the cells in the entire well condensed into a film visible to the naked eye. After the film was removed, the number of remaining adherent cells was very small. Since the cleaning of the cells was only performed according to the instructions of the separation medium, the cells obtained by centrifugation after directly adding the separation medium were mixed with twi...

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Abstract

The invention provides a method for optimally isolating monocytes in bovine peripheral blood. ACD-A is taken as an anticoagulant, the method comprises the optimization of centrifugal conditions, the optimization of cell cleaning conditions, the optimization of wall-sticking conditions and the like, and the success rate of the isolation is greatly improved. In the method, a whole set of complete system which comprises the sampling of bovine blood, the isolation of the monocytes in the bovine peripheral blood and the final transformation of the monocytes into macrophages is established for the first time, and the counting and dyeing are not required in the process, so that the time is saved and an isolation effect is improved. The survival rate of the monocytes isolated by the method is high; and the method is high in experimental repeatability and strong in operability, and provides powerful technical support for simply, conveniently and efficiently isolating the monocytes in the bovine peripheral blood.

Description

technical field [0001] The invention relates to cell separation technology, in particular to a method for separating bovine peripheral blood mononuclear cells. Background technique [0002] The isolation of peripheral blood mononuclear cells generally includes two steps: the isolation of peripheral blood mononuclear cells and the subsequent isolation of mononuclear cells. Among them, the first step is completed by density gradient centrifugation. By selecting a separation medium with a certain density, monocytes and lymphocytes with a lower density in the blood (the two have similar densities, collectively called peripheral blood mononuclear cells, PBMC) ) are separated from granulocytes and red blood cells with higher density; in the second step, monocytes can be separated from lymphocytes by other methods, including adsorption, immune sorting, reverse panning centrifugation and density gradient separation, etc. . Among them, the adsorption method is the most commonly use...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0786
Inventor 周向梅赵德明林敬钧王洋杨利峰尹晓敏
Owner CHINA AGRI UNIV
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