Pseudosciaena crocea head kidney macrophage separation and primary culture method and application thereof

A technology for primary culture of macrophages, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve problems that have not been reported

Inactive Publication Date: 2012-07-04
OCEAN UNIV OF CHINA
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Problems solved by technology

But so far, there are no reports on the establishment of in vitro model of large yellow croaker head kidney macrophages and its application in the field of nutritional immunology

Method used

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  • Pseudosciaena crocea head kidney macrophage separation and primary culture method and application thereof
  • Pseudosciaena crocea head kidney macrophage separation and primary culture method and application thereof
  • Pseudosciaena crocea head kidney macrophage separation and primary culture method and application thereof

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Embodiment Construction

[0015] The method for isolating and culturing the primary head and kidney macrophages of large yellow croaker of the present invention first utilizes the Percoll separation solution with a concentration gradient of 31% / 45%, horizontally centrifuges the large yellow croaker head and kidney macrophages, and cultures them at 22°C under the complex L15 medium. Wherein the Percoll separating solution is composed of 31% and 45% Percoll, and the volume percent ranges are 35-55% and 45-65% respectively. During implementation, 45% concentration gradient of Percoll is placed on the lower layer of the centrifuge tube, and then 31% Percoll is slowly placed on it with a syringe, which forms the 31% / 45% gradient Percoll separating solution of the present invention. The used L15 compound cell culture medium contains 50ml fetal bovine serum, 100,000 UI penicillin streptomycin, 50,000 UI heparin, 10mmol 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and 0.5g per liter of L15 medium. Gluc...

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Abstract

Provided is technology of external model construction of pseudosciaena crocea head kidney macrophage. The fish head kidney macrophage is horizontally and centrifugally separated by utilizing Percoll with 31% / 45% gradient and is cultured in an L15 compound culture medium under the condition that the temperature is 22 DEG C. Each liter of the culture medium contains 50ml fetal calf serum, 0.1 million UI penicillin / streptomycin, 0.05 million UI heparin, 10mmol 14-ethoxyl-piperazine ethanesulfonic acid (HEPES) and 0.5g glucose. The pH of the culture medium is 7.4. The method and the application have the advantages that (1) the proportion of the pseudosciaena crocea head kidney macrophage which is centrifugally separated by utilizing Percoll with 31% / 45% gradient in the separation cell is as high as 42%; (2) the purity of the adherence pseudosciaena crocea head kidney macrophage after liquid culture is as high as 90%; (3) the livability of the pseudosciaena crocea head kidney macrophage after one week culture is higher than 58%; and (4) a function checking experiment proves that LPS can remarkably affect the swallow function and breath eruption activity of the cultured pseudosciaena crocea head kidney macrophage.

Description

technical field [0001] The invention belongs to the technical field of cell models, and in particular relates to a method for separating and primary culturing large yellow croaker head kidney macrophages and an application thereof. Background technique [0002] Large yellow croaker (Larmichthys crocea) is a unique endemic fish species in my country, which is widely distributed in the coastal waters of China. Because of its delicate meat and delicious taste, large yellow croaker is very popular among consumers and is one of the most important seawater economic fishes in my country. Over the past ten years, the artificial breeding of large yellow croaker has developed rapidly, and the scale of aquaculture production has continued to expand. It has become one of the fish species with the largest number of seawater cage cultures in my country. However, due to the pollution of the aquaculture environment and the relative lag in aquaculture management, disease problems have becom...

Claims

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Application Information

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IPC IPC(8): C12N5/0786C12Q1/02
Inventor 李庆飞艾庆辉麦康森徐玮郑岳夫
Owner OCEAN UNIV OF CHINA
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