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A method for isolating and culturing zebrafish primary intestinal macrophages

A macrophage and zebrafish technology, applied in the field of cell separation and culture, can solve the problems of low expression, difficult separation methods and application of intestinal macrophages, etc. Effect

Active Publication Date: 2022-08-09
NANJING UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Their low expression of typical macrophage markers has made flow cytometry-based isolation methods difficult to apply to intestinal macrophages

Method used

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  • A method for isolating and culturing zebrafish primary intestinal macrophages
  • A method for isolating and culturing zebrafish primary intestinal macrophages

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Experimental program
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Effect test

Embodiment Construction

[0040] The present invention can be better understood from the following examples.

[0041] The specific steps for isolating and culturing zebrafish primary intestinal macrophages are:

[0042] (1) Take 8 healthy zebrafish and put them in sterilized culture water for 4 hours, and transfer them to sterile culture water containing 200IU / mL penicillin and 200μg / mL streptomycin sulfate for 12-16 hours;

[0043] (2) After killing the zebrafish, soak it in alcohol for 30s, transfer it to a sterile ultra-clean operating table, fix the fish body, use sterilized scissors and forceps to open the abdomen to kill the zebrafish, dissect the zebrafish, take out the intestine and rinse it in alcohol for a few seconds ;

[0044] (3) Cut the intestine longitudinally with a sterile needle, cut it into small tissue pieces of 1mm×1mm with scissors, and wash twice in PBS solution containing 10vt% FBS;

[0045] (4) Take 70 g of the small tissue pieces obtained in step (3) and grind them on a 70 μ...

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Abstract

The invention discloses a method for separating and culturing zebrafish primary intestinal macrophages. The cell suspension is prepared by a grinding method, and is pipetted with a sterile Pasteur pipette, so that the zebrafish intestinal cells can be dispersed in a short time, and the digestion is reduced. Enzyme damage to cells, increase the cell concentration in the cell suspension to prepare high-quality single-cell suspension, overcome the separation problems of zebrafish cells such as small particle size, low survival rate, and difficult dispersion; use fish organs to organize monocytes The separation solution effectively narrows the range of cell separation and excludes other cells and dead cells in the suspension; using the strong ability of macrophages to adhere to the wall, they can adhere to the wall within 4 hours, distinguish them from other monocytes, and effectively treat the intestinal tract. Macrophages were purified. Compared with the existing separation method, the grinding method used in the present invention can greatly shorten the test time, the preparation of the cell suspension can be completed within 10 minutes, the purity of the intestinal macrophages is higher, the operation is simple, and the obtained cell density is higher. , reducing cell agglomeration.

Description

technical field [0001] The invention belongs to the field of cell separation and culture, in particular to a method for separating zebrafish primary intestinal macrophages. Background technique [0002] Cell culture refers to a method of simulating the in vivo environment (sterility, suitable temperature, pH and certain nutritional conditions, etc.) in vitro, and maintaining the main structure and function, so that it can grow and reproduce normally. Animal cell culture began in the early 19th century, and the scale continued to expand in the mid-19th century. It has become a widely used technical method in the fields of biology and medicine. The advent of this technology has greatly facilitated the study of complex physiological processes that take place within tissue-specific cells, yielding information not available in full animal models. [0003] The objects of cell culture can be divided into two categories: cell lines and primary cells. The cells that are cultured imm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0786
CPCC12N5/0645C12N2509/00Y02A40/81
Inventor 吴兵顾纬卿余静吴小梅
Owner NANJING UNIV
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