Method for separating 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, reducing the requirement of experimental skills, and simplifying the effect of experimental steps

Active Publication Date: 2019-02-01
HUAZHONG AGRI UNIV
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Problems solved by technology

So far, there is no method for isolating

Method used

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  • Method for separating and primary culture of grass carp dendritic cells
  • Method for separating and primary culture of grass carp dendritic cells
  • Method for separating and primary culture of grass carp dendritic cells

Examples

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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 make statistics. The purity of the grass carp dendritic cells can reach as high as 80 %above.

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Abstract

The invention provides a method for separating and primary culture of grass carp dendritic cells, which comprises the following steps: step 1. separation of grass carp spleen and head kidney: anesthetizing grass carp to death, removing spleen and head kidney, and removing adherent connective tissues and adipose tissues and performing immersion; step 2: preparation of grass spleen and head kidney single cell suspension: performing continuous mesh filtration to obtain the cell suspension; step 3. separation of grass spleen and head kidney mononuclear cells: performing Ficoll separation, after centrifugation, and taking the cells of a white film layer and washing the material; step 4. static culture: adjusting the concentration of monocytes and culturing the material in an incubator; and step5. enriching and purifying of the dendritic cells of grass carp: collecting the suspension cells after a period of culture, and purifying the dendritic cells of the grass carp by a density gradient centrifugation method. The method can effectively prepare a large number of grass carp dendritic cells, and the obtained grass carp dendritic cells have a purity of more than 80%; the operation processis simple and clear, and the time consuming 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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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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