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Culture medium for flounder paralichthys lymphocyte proliferation

A lymphocyte proliferation and lymphocyte technology, applied in the field of culture medium preparation, to achieve the effect of high lymphocyte viability index and good stability

Inactive Publication Date: 2016-01-20
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

How to obtain high-efficiency fish lymphocyte proliferation medium remains unsolved

Method used

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  • Culture medium for flounder paralichthys lymphocyte proliferation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: the preparation of culture medium

[0023] The culture medium of the present embodiment, its preparation steps are as follows:

[0024] 1) Under aseptic conditions, weigh the dry powder of DMEM medium on an electronic balance and place it in a sterile container, add sterile water to dilute, stir and mix to obtain a DMEM liquid medium;

[0025] 2) Add concanavalin A (ConA) to the medium, and the final concentration added is 20ug / ml;

[0026] 3) adding bacterial lipopolysaccharide (LPS) to the culture medium, the final concentration added is 100ug / ml;

[0027] 4) Adding phytohemagglutinin (PHA-P) into the culture medium, the final concentration added is 2ug / ml;

[0028] 5) Pokeweed agglutinin (PWM) was added to the medium, and the final concentration added was 5ug / ml;

[0029] 6) adding mixed deoxymononucleotides to the culture medium, the final concentration added is 10mmol / L;

[0030] 7) Sodium bicarbonate is added to the culture medium, and the final ...

Embodiment 2

[0041] Embodiment 2, the cultivation of culture medium of the present invention

[0042] The medium of the present invention prepared by using the examples; the formula of the comparison medium: L15 or 199; 89.9%, fetal bovine serum 10%, double antibody 0.1%.

[0043] Lymphocyte culture method: extract 2ml of flounder fish blood under sterile conditions, add an equal volume of FICOLL lymphocyte separation medium, centrifuge at 24°C and 2000rcf for 1 hour, suck out the middle lymphocyte layer, wash the lymphocytes with PBS, add medium, place in Cultivate in a 24°C incubator for 72 hours, and gently shake the culture dish every 9 hours or so to promote cell proliferation and growth.

[0044] Chromosome preparation: conventional chromosome preparation techniques were used for chromosome preparation, including colchicine treatment for 2 hours, potassium chloride hypotonic treatment for 30 minutes, Carnot's fixative pre-fixation for 10 minutes, and continued fixation for 3 times, e...

Embodiment 3

[0046] Example 3, detection of lymphocyte proliferation activity of flounder and flounder

[0047] The culture medium of the present invention is used.

[0048] Contrast medium formula: L15 or 19989.9%, fetal bovine serum 10%, double antibody 0.1%.

[0049] Lymphocyte culture method: extract 2ml of flounder fish blood under sterile conditions, add an equal volume of FICOLL lymphocyte separation medium, centrifuge at 24°C and 2000rcf for 1 hour, suck out the middle lymphocyte layer, wash the lymphocytes with PBS, add the medium, and aliquot In a 96-well cell culture plate, culture in a 24°C incubator for 72 hours, and gently shake the culture dish once every 9 hours or so to promote cell proliferation and growth.

[0050] Detection of lymphocyte proliferation activity: After 72 hours of culture, add 10 μl of cell proliferation detection reagent CCK-8 to each well, continue to culture at 24°C for 3 hours, take out the 96-well cell culture plate, and observe the color. The darke...

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Abstract

The invention provides a culture medium for flounder paralichthys lymphocyte proliferation. The culture medium is formed by adding concanavalin A, lipopolysaccharide, phytohemagglutinin, America pokeweed lectins, flounder paralichthys serum, fetal calf serum, mixed deoxygenation mononucleotide, L-glutamine and sodium bicarbonate into a basic culture medium. The culture medium contains the flounder paralichthys serum and various types of mitogen for promoting proliferation of lymphocyte, through effective matching of all ingredients, efficient and rapid proliferation of the lymphocyte can be achieved, the activity index of the lymphocyte is high, the average proliferation activity is as high as 85.33%, and meanwhile the culture medium has good stability and can completely meet the requirement of culturing lymphocyte of the fish molecular immunology and the cytogenetics.

Description

technical field [0001] The invention belongs to the technical field of medium preparation, and in particular relates to a medium for proliferation of flounder and flounder lymphocytes. Background technique [0002] Fish is known as the vertebrate with the lowest level of innate and acquired immunity, and also has a complete humoral and cellular immune response. As the main cell type in fish blood immunity, lymphocytes play a very important role in the overall immune system of fish. [0003] The mass proliferation and culture of fish lymphocytes in vitro not only provides sufficient materials for traditional fish cytogenetic research, but also lays the foundation for current fish molecular immunology research. [0004] In order to efficiently and massively proliferate fish lymphocytes in vitro, the composition of the medium is very important. The traditional basal medium provides basic conditions for the survival of fish lymphocytes, including suitable PH value and osmotic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
Inventor 王旭波李赞刘秀梅张全启齐洁王志刚于海洋贺艳
Owner OCEAN UNIV OF CHINA
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