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A method for separating blood cells of Portunus trituberculatus

A technology of portunus trituratus and a separation method, which is applied to cell dissociation methods, animal cells, invertebrate cells, etc., can solve the problem of not developing crab blood cell separation technology, etc., and achieves simple operation, high separation efficiency, and good activity. Effect

Active Publication Date: 2021-03-02
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no technical report on the complete separation of the three blood cells by one-step density gradient centrifugation at home and abroad, and there is no research on the separation technology of crab blood cells at home and abroad.

Method used

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  • A method for separating blood cells of Portunus trituberculatus
  • A method for separating blood cells of Portunus trituberculatus
  • A method for separating blood cells of Portunus trituberculatus

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Effect test

Embodiment 1

[0035] A kind of Portunus trituberculatus blood cell separation method that present embodiment proposes, it comprises the following steps:

[0036] Step 1, preparation of cell separation solution:

[0037] 1_1. Prepare a PBS buffer solution with a volume molar concentration of 0.1M.

[0038] 1_2. Using a sodium chloride solution with a volume molar concentration of 3M to adjust the osmotic pressure of the PBS buffer solution to 900 mOs·mol / kg to obtain an isotonic solution. Among them, 10ml of PBS buffer solution and 1.2ml of sodium chloride solution can be taken.

[0039] 1-3, the isotonic solution is used as diluent, and the volume percent concentration of dilution is 60% iodixanol (OptiPrep TM ), three kinds of iodixanol solutions with volume percentage concentration of 10%, 15% and 20% were configured in gradient.

[0040] 1_4. Using the existing cushion technology, add three kinds of iodixanol solutions with volume percentage concentrations of 10%, 15% and 20% respecti...

Embodiment 2

[0054] This embodiment carries out further processing on the basis of the first embodiment. That is: after step 3_2, the clear cell solution in the separated clear cell layer, the small granular cell solution in the small granular cell layer, and the large granular cell solution in the large granular cell layer are washed with cell separation liquid to keep more Good activity, the specific process is as follows: absorb the clear cell solution from the clear cell layer, add isotonic solution whose volume is 3 times the volume of the absorbed clear cell solution, and centrifuge at 1200g for 2.5 minutes at 4°C , to remove the supernatant to obtain a precipitate; then add an isotonic solution with a volume 3 times the volume of the precipitate to the precipitate, perform centrifugation at 1200g for 2.5min at 4°C, remove the supernatant, and obtain an elution The clear cell solution after the cell separation medium; in the same manner, the small granular cell solution after the cel...

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Abstract

The invention discloses a blood cell separation method for portunus trituberculatus. The method comprises the following steps: diluting iodixanol with an isotonic solution obtained by regulating an osmotic pressure of a PBS buffer solution by utilizing a sodium chloride solution, and performing gradient preparation to obtain three iodixanol solutions of different concentrations; forming gradient solutions of different densities by adopting a cushion technology, and standing to form a continuous density gradient solution to serve as a cell separation solution; collecting blood cells of portunustrituberculatus, and taking mixed cell suspension obtained by diluting the isotonic solution; slowly dropping the mixed cell suspension into the cell separation solution, centrifuging, and dividing the blood cells of portunus trituberculatus into three layers, wherein the uppermost layer refers to clear cells, the intermediate layer refers to small granular cells, and the lowest layer refers to large granular cells. The method has the advantages that only one-step density gradient centrifugation is adopted, the operation is simple, and experiments discover that the separated blood cells haveexcellent viabilities and can be used for cell culture.

Description

technical field [0001] The invention relates to a cell separation technology, in particular to a method for separating blood cells of Portunus trituberculatus. Background technique [0002] Portunus trituberculatus, commonly known as white crab and gun crab, belongs to the subphylum Crustacea, class Malacostraca, order Decapoda, family Portunidae, and the genus Portunus. Widely distributed in sea areas such as China, Japan, North Korea and the Malaysian archipelago, it is an important seafood economic crab in my country. In recent years, Portunus trituberculatus diseases have occurred frequently. According to statistics, among various diseases, pathogenic diseases such as bacteria, viruses and parasites are the main ones, which have caused great losses to my country's aquaculture industry and coastal economy. At present, basic research on the innate immunity of marine crustaceans is gradually increasing to speed up the process of immune prevention and treatment of invertebra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/07
CPCC12N5/0601C12N2509/00
Inventor 周素明杨宁王国良陶震
Owner NINGBO UNIV
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