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A kit for isolating and culturing macrophages derived from livestock peripheral blood
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A technology of macrophages and peripheral blood, which is applied in the field of kits for the isolation and cultivation of macrophages from peripheral blood of livestock, and can solve problems such as difficulty in long-term survival
Active Publication Date: 2021-07-13
CHINA AGRI UNIV
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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
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Embodiment 1
[0042] Example 1, composition for separating and culture macrophages
[0043] The kit is composed of a single nuclear cell separation component and macrophage culture component;
[0044] Single nuclear cell separation components include Hank's solution, PBS solution, and human peripheral blood lymphocyte separation;
[0045] Macrophage culture components include macrophage culture fluids;
[0046] Macrophage culture fluid consists of fetal bovine serum and RPMI-1640 medium; the concentration of fetal bovine serum in macrophage culture fluid is 10% (volume percent).
Embodiment 2
[0047] Example 2, Separation, Culture and Identification of Sheep Peripheral Blood Macrophage
[0048] First, preparation of single nuclear cells
[0049] 1. Uniformly mixed 1 body of the cervical venous blood and 1 volume of Hank's solution to obtain a mixed solution.
[0050] 2, a mixed solution obtained by 1 volume of step 1 is added to an outer peripheral blood lymphocyte separation solution, 2000 r / min is centrifuged for 20 min, and the intermediate white haze layer is transferred to a centrifuge tube, 2000 r / min centrifuge for 5 min Collect precipitate.
[0051] 3. The cell precipitate obtained by resuspending step 2 using a 3 ml Hank's solution, and the cell precipitation is collected (ie peripheral blood mononuclear cells).
[0052] Second, the preparation of macrophages
[0053] 1. Use the lymphocyte precipitate obtained by the culture of the macrophage cell culture solution to 10 6 The concentration of cells / ml inoculated in 6 wells, placed at 37 ° C, 5% CO 2 The c...
Embodiment 3
[0073] Example 3 Separation, Culture and Identification of Shotophyte Blood Macrophages
[0074] First, preparation of single nuclear cells
[0075] 1. Uniformly mixed 1 volume of pork neck venous blood and 1 volume of Hank's solution to obtain a mixed solution.
[0076] 2, a mixed solution obtained by 1 volume of step 1 is added to an outer peripheral blood lymphocyte separation solution, 2000 r / min is centrifuged for 20 min, and the intermediate white haze layer is transferred to a centrifuge tube, 2000 r / min centrifuge for 5 min Collect precipitate.
[0077] 3. The cell precipitate obtained by resuspending step 2 using a 3 ml Hank's solution, and the cell precipitation is collected (ie peripheral blood mononuclear cells).
[0078] Second, the preparation of macrophages
[0079] 1. Use the lymphocyte precipitate obtained by the culture of the macrophage cell culture solution to 10 6 The concentration of cells / ml inoculated in 6 wells, placed at 37 ° C, 5% CO 2 The carbon d...
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Abstract
The invention discloses a kit for isolating and culturing macrophages derived from livestock peripheral blood. The invention protects a kit for preparing macrophages, including Hank's solution, peripheral blood lymphocyte separation liquid and macrophage culture liquid. The present invention centrifuges the mixture of jugular venous blood, Hank's solution, and human peripheral blood lymphocyte separation liquid (1:1:2 uniform mixing) by blood density gradient centrifugation to obtain peripheral blood mononuclear cells, which are separated and cultured in vitro 7‑10 days to obtain macrophages. Experiments have proved that the macrophages isolated and cultivated by the kit of the present invention have spontaneous proliferation and phagocytosis capabilities, and can be subcultured for more than 20 times. The macrophages isolated and cultured are morphologically consistent with those cultivated by traditional methods; The identification of the phagocytosis ability of the phagocytic cells is not much different from the cells cultivated by the traditional method, and the preparation of the kit of the invention is simple and the cost is low.
Description
Technical field [0001] The present invention relates to a kit for separating and culturing a source of macrophages induced by external livestock. Background technique [0002] The high incidence of animal diseases is a major challenge facing the development of animal husbandry. According to statistics, 12% to 15% of the total output value of animal husbandry every year is caused by livestock diseases. The genetic method is used to improve the animal's immune function from genetics, increasing the effectiveness of the animal's resistance to disease. With the development of cell molecular biology, the process of animal disease-resistant breeding operation was accelerated by the development of immune cells. [0003] Macrophages are one of the important immune cells of mammalian body, involved in inherent immune responses, to swallowing digestive function on pathogenic microorganisms, with antigen processing presence, participation in adaptive immune response, in the body resistance ...
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