A method for the isolation and primary culture of grass carp dendritic cells

A dendritic cell and primary culture technology, which is applied in the field of grass carp dendritic cell isolation and primary culture, can solve problems such as the method of isolating and culturing grass carp dendritic cells that have not yet been seen, and reduce the risk of cell contamination Probability, less damage to the donor, and easy access to the effect

Active Publication Date: 2021-11-09
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there is no method for isolating and culturing grass carp dendritic cells

Method used

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  • A method for the isolation and primary culture of grass carp dendritic cells
  • A method for the isolation and primary culture of grass carp dendritic cells
  • A method for the isolation and primary culture of grass carp dendritic cells

Examples

Experimental program
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Embodiment 1

[0034] 1. Preparation of experimental materials

[0035] The composition and proportion of the culture medium or culture medium used in the experiment are as follows

[0036] AIM solution: 47.50ml L-15 medium, 2.50ml double antibody, 2.5μg / mL amphotericin B and 25μg / mL gentamicin, put at 4℃ for later use;

[0037] Primary cell culture medium: 42.50ml L-15 medium, 5mL FBS, 2.50ml double antibody, 2.5μg / mL amphotericin B and 25μg / mL gentamicin, store at 4°C for later use;

[0038] 2. Experimental method

[0039] 1. Separation of spleen and head kidney of grass carp: anesthetize grass carp to death with excessive MS-222. The body surface was sprayed with 75% alcohol and repeatedly wiped and disinfected with alcohol cotton balls, then transferred to a sterile ultra-clean bench, and blood was collected from the tail vein.

[0040] Separation of the spleen: Use sterile surgical scissors to open the abdominal cavity along the lateral line of the fish body, and remove the spleen wi...

experiment example

[0047] After the grass carp dendritic cells obtained in the above examples were enriched and purified, they were placed on a flow cytometer to detect the purity of the cells. In flow cytometry, forward scattered light (Forward scatter, FSC) reflects the size of the cell, side scattered light (Side scatter, SSC) reflects the complexity of the cell, the size and complexity of different types of cells The difference is large, so they can be divided into groups. Therefore, FSC and SSC of flow cytometry were used here to analyze the purity of dendritic cells after enrichment. Experimental results such as Figure 7 As shown, the whole cells are concentrated into a group, and there are very few cells outside the group, indicating that the obtained grass carp dendritic cells are of high purity, and the statistical function of the flow cytometer is used to calculate the purity of the grass carp dendritic cells as high as 80% %above.

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Abstract

The present invention provides a method for the isolation and primary culture of grass carp dendritic cells, comprising the following steps: Step 1, the separation of grass carp spleen and head kidney: the grass carp is anesthetized to death, the spleen and head kidney are taken out, and the attached connective tissue is removed and adipose tissue and soaked; step 2, preparation of grass carp spleen and head kidney single cell suspension: continuous sieve filtration to obtain cell suspension; step 3, separation of grass carp spleen and head kidney mononuclear cells: Ficoll separation, after centrifugation Absorb and wash the cells of the buffy coat; step 4, static culture: adjust the concentration of monocytes, and place them in an incubator for culture; step 5, enrichment and purification of grass carp dendritic cells: collect suspension cells after culturing for a period of time, and use Grass carp dendritic cells were purified by density gradient centrifugation. The method can effectively prepare a large amount of grass carp dendritic cells, and the purity of the obtained grass carp dendritic cells can reach more than 80 percent; the operation process is simple and clear, and the time consumption is short.

Description

technical field [0001] The invention relates to the technical field of in vitro culture of fish cells, in particular to a method for the isolation and primary culture of grass carp dendritic cells. Background technique [0002] As an important technology of cell biology and even biological research, cell culture occupies an important position in the field of biological research. Animal tissue (cell) culture began in the early 20th century, and has become a widely used technique in biological and medical research and applications. Fish cell culture started relatively late, since the 1960s. Mainly used in virology, immunology and physiological and toxicology research. The isolation and culture of fish dendritic cells is of great significance to the prevention, diagnosis and treatment of fish diseases. [0003] Dendritic cell (DC) is the most powerful antigen presenting cell (Antigen presenting cell, APC) known in vivo (Steinman, 1991). DC is a key factor in identifying inte...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0784
CPCC12N5/0639C12N2509/10
Inventor 陈孝煊周成翀吴志新李思思王辉郭道远
Owner HUAZHONG AGRI UNIV
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