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Kit for separating and culturing PB (Peripheral Blood) derived macrophagus of livestock

A technology for macrophages and peripheral blood, applied in the field of kits for the isolation and cultivation of macrophages derived from peripheral blood of livestock, can solve problems such as difficulty in long-term survival, and achieve the effect of low cost and simple preparation

Active Publication Date: 2018-01-26
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Macrophages can live for 2-3 weeks under suitable conditions, and are mostly used for primary culture, and it is difficult to survive for a long time

Method used

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  • Kit for separating and culturing PB (Peripheral Blood) derived macrophagus of livestock
  • Kit for separating and culturing PB (Peripheral Blood) derived macrophagus of livestock
  • Kit for separating and culturing PB (Peripheral Blood) derived macrophagus of livestock

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1. Composition of the kit for isolating and culturing macrophages

[0043] The kit consists of mononuclear cell isolation components and macrophage culture components;

[0044] Mononuclear cell separation components include Hank's solution, PBS solution and human peripheral blood lymphocyte separation medium;

[0045] The macrophage culture components include macrophage culture fluid;

[0046] The macrophage culture medium is composed of fetal bovine serum and RPMI-1640 medium; the concentration of fetal bovine serum in the macrophage culture medium is 10% (volume percentage).

Embodiment 2

[0047] Example 2. Isolation, cultivation and identification of sheep peripheral blood macrophages

[0048] 1. Preparation of mononuclear cells

[0049] 1. Uniformly mix 1 volume part of sheep jugular vein blood and 1 volume part of Hank's solution to obtain a mixed solution.

[0050] 2. Add 1 volume part of the mixed solution obtained in step 1 to 1 volume part of human peripheral blood lymphocyte separation medium, centrifuge at 2000r / min for 20min, transfer the cells in the middle white misty layer to a centrifuge tube, and centrifuge at 2000r / min for 5min , to collect the precipitate.

[0051] 3. Use 3ml Hank's solution to resuspend the cell pellet obtained in step 2, repeat washing twice, and collect the cell pellet (ie peripheral blood mononuclear cells).

[0052] 2. Preparation of macrophages

[0053] 1. Use macrophage cell culture medium to resuspend the lymphocyte pellet obtained in step 1, and use 10 6 The concentration of cells / ml was inoculated in 6-well culture...

Embodiment 3

[0073] Example 3, Isolation, Culture and Identification of Porcine Peripheral Blood Macrophages

[0074] 1. Preparation of mononuclear cells

[0075] 1. Uniformly mix 1 volume part of porcine jugular vein blood and 1 volume part of Hank's solution to obtain a mixed solution.

[0076] 2. Add 1 volume part of the mixed solution obtained in step 1 to 1 volume part of human peripheral blood lymphocyte separation medium, centrifuge at 2000r / min for 20min, transfer the cells in the middle white misty layer to a centrifuge tube, and centrifuge at 2000r / min for 5min , to collect the precipitate.

[0077] 3. Use 3ml Hank's solution to resuspend the cell pellet obtained in step 2, repeat washing twice, and collect the cell pellet (ie peripheral blood mononuclear cells).

[0078] 2. Preparation of macrophages

[0079] 1. Use macrophage cell culture medium to resuspend the lymphocyte pellet obtained in step 1, and use 10 6 The concentration of cells / ml was inoculated in 6-well culture...

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Abstract

The invention discloses a kit for separating and culturing a PB (Peripheral Blood) derived macrophagus of livestock. The kit for preparing the macropagus comprises a Hank's solution, a PB lymphocyte separating solution and a macrophagus separating solution. According to the kit disclosed by the invention, a mixed solution of jugular venous blood, the Hank's solution and the human PB lymphocyte separating solution (which are uniformly mixed in a ratio of 1 to 1 to 2) can be centrifuged through a blood density gradient centrifugation method, PB mononuclear cell is obtained, and the macrophagus can be obtained by carrying out in-vitro separating and culturing for 7 to 10 days. An experiment verifies that the macrophagus separated and cultured by the kit has spontaneous proliferation and phagocytosis abilities and can be subjected to 20 times of subculture or above, and the separated and cultured macrophagus is consistent with macrophagus cultivated by a traditional method in morphology; authentication on the pahgocytosis ability of the macrophagus is not quite different from that of cell cultivated by the traditional method; the kit disclosed by the invention is simple in preparationand low in cost.

Description

technical field [0001] The invention relates to a kit for isolating and culturing macrophages derived from livestock peripheral blood. Background technique [0002] The high incidence of animal diseases is a major challenge to the current development of animal husbandry. According to statistics, 12% to 15% of the total output value of animal husbandry is lost every year due to diseases of livestock and poultry. The use of genetic methods to improve the immune function of animals in essence and increase the resistance of animals to diseases has the effect of curing the root cause. With the development of cell and molecular biology, the process of animal disease-resistant breeding has been accelerated through the study of the immune mechanism of immune cells. [0003] As one of the important immune cells in mammals, macrophages participate in the innate immune response, have the function of phagocytosis and digestion of pathogenic microorganisms, have the function of antigen ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0786
Inventor 韩红兵刘永晓刘哲熹连正兴
Owner CHINA AGRI UNIV