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Method of activating T lymphocyte of mouse in vitro

A lymphocyte and mouse technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of low activation rate and long activation time, and improve activation efficiency and accuracy. good, fast effect

Inactive Publication Date: 2018-11-20
THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, simple T cell activation takes 20 to 48 hours, the time required for activation is too long, and the activation rate is not very high.

Method used

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  • Method of activating T lymphocyte of mouse in vitro
  • Method of activating T lymphocyte of mouse in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A method for activating mouse T lymphocytes in vitro, the specific steps are as follows:

[0044] A. A 7-week-old clean-grade mouse was killed by neck dislocation, soaked in 75% alcohol solution for 5 minutes, aseptically removed the spleen, and placed it in a diameter medium containing 18 mL of phosphate buffer and 2 mL of fetal calf serum. In a 10cm sterile petri dish;

[0045] B. Grinding the mouse spleen obtained in step A with the rough surface of a sterile frosted glass slide until homogenized;

[0046] C. Use a cell mesh with a pore size of 70 μm to filter the homogenate obtained in step B, then use a sterile 15 mL conical centrifuge tube to collect the filtrate, and centrifuge at 300 × g for 5 minutes, discard the supernatant to collect the cell pellet;

[0047] D. Add 3 mL of sterile erythrocyte lysate to the centrifuge tube to resuspend the cells and incubate at room temperature for 3-5 minutes;

[0048] E. Add 10 mL of sterile phosphate buffer solution to the...

Embodiment 2

[0063] A method for activating mouse T lymphocytes in vitro, the specific steps are as follows:

[0064] A. A 7-week-old clean-grade mouse was killed by neck dislocation, soaked in 75% alcohol solution for 6 minutes, aseptically removed the spleen, and placed it in a diameter of In a 10cm sterile petri dish;

[0065] B. Grinding the mouse spleen obtained in step A with the rough surface of a frosted glass slide until homogenized;

[0066] C. Use a cell mesh with a pore size of 70 μm to filter the homogenate obtained in step B, then use a sterile 15 mL conical centrifuge tube to collect the filtrate, and centrifuge at 300 × g for 5 minutes, discard the supernatant to collect the cell pellet;

[0067] D. Add 3 mL of sterile erythrocyte lysate to the centrifuge tube to resuspend the cells and incubate at room temperature for 3-5 minutes;

[0068] E. Add 10 mL of sterile phosphate buffered saline to the centrifuge tube and mix well. Centrifuge at 300×g for 5 minutes at room tem...

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Abstract

The invention belongs to the biotechnical field and in particular relates to a method of activating a T lymphocyte of a mouse in vitro. The method specifically comprises the following steps: A, acquiring lymphocytes of a spleen, peripheral lymph nodes, mucous epithelium or peripheral blood of the mouse; B, screening CD3 epsilon+lymphocyte and detecting the purity; C, coating a 48 porous cell culture plate with anti-CD3 epsilon and CD28 antibodies of the mouse with bioactivity; D, adding a culture medium resuspended cell of the activated T lymphocyte into the the cell culture plate obtained inthe step C; and E, culturing the cell plate obtained in the step D in a CO2 culture box for 8-12 hours. According to the method, the activating rate of the T lymphocyte reaches 90% or over within a short time, and the method has important meaning in follow-up researches of the T lymphocyte in various biological function aspects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for activating mouse T lymphocytes in vitro. Background technique [0002] T lymphocytes originate from the pluripotent stem cells of the bone marrow (from the yolk sac and liver during the embryonic period). During the embryonic and nascent stages of the human body, a part of the pluripotent stem cells or pre-T cells in the bone marrow migrate into the thymus. Differentiate and mature to become immunocompetent T cells. Mature T cells are distributed through the bloodstream to settle in the thymus-dependent areas of peripheral immune organs, and can be recirculated through lymphatic vessels, peripheral blood, and tissue fluid to exert cellular immunity and immune regulation functions. The recycling of T cells is conducive to extensive exposure to antigenic substances entering the body, strengthening the immune response, and maintaining immune memory for a long t...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2500/12C12N2500/30C12N2500/32C12N2500/44C12N2501/2302C12N2501/51C12N2501/515
Inventor 杨加彩刘美希王旭李传贵宋亚军
Owner THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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