Flow cytometry detection method of nitric oxide level of shrimp blood corpuscles

A flow cytometry and nitric oxide technology, which is applied in the field of flow cytometry detection of nitric oxide content in shrimp blood cells, can solve the problems of difficulty in analyzing the nitric oxide content of different types of cells, inability to reflect the content, and poor repeatability. , to achieve the effect of avoiding operation damage, good repeatability and high accuracy

Inactive Publication Date: 2013-04-24
SOUTH CHINA NORMAL UNIVERSITY
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  • Abstract
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  • Claims
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Problems solved by technology

This method has the following obvious shortcomings and deficiencies: 1. Not all nitrite and nitrate in the body are derived from the conversion of nitric oxide, and the repeatability is poor; 2. This meth

Method used

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  • Flow cytometry detection method of nitric oxide level of shrimp blood corpuscles
  • Flow cytometry detection method of nitric oxide level of shrimp blood corpuscles
  • Flow cytometry detection method of nitric oxide level of shrimp blood corpuscles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Use flow cytometry to determine the nitric oxide content of different types of blood cells of Penaeus monodon, including the following steps:

[0027] (1) Preparation of anticoagulant suitable for Penaeus monodon: add glucose 20.5g / L, sodium citrate 8g / L, sodium chloride 4.2g / L in distilled water, adjust the pH to 7.5, autoclave, and cool it. Store in a refrigerator at 4°C for later use.

[0028] (2) Prepare the blood cell suspension of prawns: take out prawns, wipe dry body surface water with cotton balls, and use sterile syringes pre-extracted with sterilized and pre-cooled anticoagulant to draw the prawns from the pericardial cavity or abdominal sinus. Anticoagulant equal volume of hemolymph, use pre-chilled anticoagulant to adjust the cell concentration to about 10 6 cells / mL, a total of 15 prawns were taken, and the blood cell suspension of each prawn was used as a separate sample for determination.

[0029] (3) Co-incubation of fluorescent dye DAF-FM DA with blood cells...

Embodiment 2

[0034] Use flow cytometry to determine the content of nitric oxide in isolated blood cells of Penaeus monodon treated with lipopolysaccharide, including the following steps:

[0035] (1) Preparation of anticoagulant suitable for Penaeus monodon: add glucose 20.5g / L, sodium citrate 8g / L, sodium chloride 4.2g / L in distilled water, adjust the pH to 7.5, autoclave, and cool it. Store in a refrigerator at 4°C for later use.

[0036] (2) Prepare the blood cell suspension of prawns: take out prawns, wipe dry body surface water with cotton balls, and use sterile syringes pre-extracted with sterilized and pre-cooled anticoagulant to draw the prawns from the pericardial cavity or abdominal sinus. Anticoagulant equal volume of hemolymph, put it in a sterile centrifuge tube, take enough hemolymph, mix it, adjust the cell concentration to about 10 with pre-chilled anticoagulant 6 cells / mL.

[0037] (3) Lipopolysaccharide (LPS) treatment of blood cells: Lipopolysaccharide (LPS) (Escherichia coli0...

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Abstract

The invention discloses a flow cytometry detection method of nitric oxide level of shrimp blood corpuscles. The flow cytometry detection method comprises the following steps of preparing shrimp blood corpuscle suspension liquid, co-culturing fluorochromes DAF-FM DA and shrimps blood corpuscles, and measuring DAF-FM average flourescence intensity of shrimp total blood corpuscles or blood corpuscles of different types through flow cytometry. The flow cytometry detection method is free from cell washing, avoids operation damage on the blood corpuscles, is high in accuracy, good in repeatability, large in measured quantity, simple, convenient and rapid to operate, and capable of analyzing cell nitric oxide level of different types at the same time, and provides a method basis for measuring the nitric oxide level of the shrimp blood corpuscles.

Description

Technical field [0001] The invention belongs to the technical field of marine biology, and specifically relates to a flow cytometry method for detecting the content of nitric oxide in shrimp blood cells. Background technique [0002] Shrimp is an important agricultural product for the structural adjustment of aquaculture in my country, the increase of farmers' production and income, and the country's export earnings. However, in recent years, large-scale illnesses and deaths have frequently occurred in shrimp farming, causing serious economic losses. Shrimp immunology research can understand the characteristics of the immune response of shrimps, so as to lay the theoretical foundation and scientific basis for the nutritional immunity and disease prevention of shrimps. [0003] In recent years, a variety of shrimp nitric oxide synthase gene sequences have been cloned one after another. Research suggests that nitric oxide also plays an important role in the immune defense of shrimps...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 王安利冼健安苗玉涛潘训彬李彬郭慧张胜鹏
Owner SOUTH CHINA NORMAL UNIVERSITY
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