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Method used for detecting ostrea plicatula eosinophilic and basophilic granulocyte phagocytic ability

A technology of basophils and pleated oysters, which is applied in the field of detection of eosinophilic and basophilic phagocytosis of pleated oysters, can solve the problems of low repeatability, long time-consuming, poor specificity, etc., and achieve high repeatability and operation Simple, Specific Effects

Inactive Publication Date: 2014-08-20
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

Although fluorescent microspheres have the characteristics of uniform particle size, strong specificity, and high stability; The size (cell size, FSC) and cytoplasmic granularity (cytoplasmic granularity, SSC) make it difficult to clearly distinguish these two types of granulocytes in the flow cytometry scattergram, that is, the flow cytometer detects these two types of granulocytes. There are limitations in the phagocytosis of granulocytes
Although the Giemsa staining method can clearly distinguish eosinophilic (cytoplasmic red) and basophilic (cytoplasmic blue) granulocytes, the bacteria engulfed in the cytoplasm are also very easy to stain, resulting in confusion with cytoplasmic granules , poor specificity; additional culture of inactivated bacteria is required, which takes a long time and has low repeatability

Method used

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  • Method used for detecting ostrea plicatula eosinophilic and basophilic granulocyte phagocytic ability
  • Method used for detecting ostrea plicatula eosinophilic and basophilic granulocyte phagocytic ability

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Embodiment 1

[0025] (1) Preparation of neutral red staining solution: Use phosphate buffered saline (PBS: 8.0 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate, 1000 ml distilled water with pH 7.0 , adjust the pH value to 7.0 with HCl or NaOH) Dissolve the commercially available neutral red powder (Shanghai Reagent No. save. When in use, dilute the stock solution 10 times with PBS, and prepare it now.

[0026] (2) Preparation of pleated oyster blood cell suspension: take pleated oysters, use 5ml containing 2ml pre-cooled anticoagulant (glucose 20.8g, sodium citrate 8.0g, EDTA 3.36g, sodium chloride 22.3g, distilled water 1000ml, pH7. 2) The syringe is used to extract hemolymph from the adductor muscle, each 2ml. The extracted hemolymph was centrifuged at 3000r / min at 4°C for 10min, and the resulting blood cell pellet was resuspended in PBS. Hemocytometer adjusted cell concentration to 10 6 cells / ml.

[0027] (3) Fluore...

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Abstract

The invention relates to a method used for detecting ostrea plicatula eosinophilic and basophilic granulocyte phagocytic ability, and the method comprises (1) ostrea plicatula blood cell suspension preparation; (2) fluorescent microsphere phagocytosis; (3) staining with neutral red; (4) lining dying with propidium iodide; and (5) microscopy. The method has the advantages of being simple in operation, rapid and accurate, strong in specificity, high in repeatability, and the like.

Description

technical field [0001] The invention belongs to the field of pleated oyster cell immunology, in particular to a method for detecting the phagocytosis of eosinophilic and basophilic granulocytes of pleated oyster. Background technique [0002] Pleated oyster (Ostrea plicatula) is the main breeding object in the north and south coasts of my country. In recent years, with the aggravation of the aquaculture scale and the deterioration of the aquaculture environment, problems such as weakened resistance to stress, decreased immunity, and susceptibility to disease have gradually appeared. There are still no feasible solutions to these problems, and the main reason is that the understanding of the immune defense mechanism of shellfish is not very clear and thorough. Therefore, it is urgent to carry out basic research on shellfish immunology. [0003] Due to the lack of specific immunity mediated by immunoglobulins, the immune defense mechanism of shellfish mainly relies on non-spe...

Claims

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Application Information

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IPC IPC(8): G01N15/10G01N1/28
Inventor 林听听周凯张东来琦芳么宗利
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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