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Separation method of shark mononuclear cells, shark diluent and application thereof

A technology of nucleated cells and diluents, applied in the field of cell separation, can solve the problems of inability to separate the mononuclear cells of the striped bamboo shark and the high proportion of living cells, achieve high activity and purity, and reduce pollution

Active Publication Date: 2022-06-24
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem that the existing peripheral blood mononuclear cell separation technology cannot realize the separation of mononuclear cells of the striped bamboo shark, and to provide a diluent and method suitable for the separation of mononuclear cells of the striped bamboo shark, which can separate the striped bamboo shark. The mononuclear cells of the bamboo shark were separated from the red blood cells and granulocytes, and the proportion of living cells in the isolated mononuclear cells was high

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  • Separation method of shark mononuclear cells, shark diluent and application thereof
  • Separation method of shark mononuclear cells, shark diluent and application thereof
  • Separation method of shark mononuclear cells, shark diluent and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Dilution and treatment before the separation of peripheral blood cells of the striped bamboo shark

[0036] In order to obtain a good separation effect, the blood needs to be properly anticoagulated and diluted according to the following steps after collection to ensure that the blood cells are in a single dispersed state.

[0037] (1) Prepare 10×PBS solution (1L): 80g NaCl, 2g KCl, 14.4g Na 2 HPO 4 , 2.4g KH 2 PO 4 , add 800 mL of ultrapure water, adjust the pH to 7.4, and finally make up to 1L with ultrapure water.

[0038] (2) Prepare shark diluent (1L): 100mL 10×PBS (PH 7.4), 100mL 2M urea, 50mL 1MNaCl, add ultrapure water to make up to 1L. Measure the density of this dilution, it should be 1.0200g / ml.

[0039](3) Obtaining fresh blood of Bamboo shark, adding anticoagulant sodium citrate solution (4%) to prevent blood coagulation, and the volume ratio of anticoagulant to blood is between 1:9-2:8. (Because the speed of blood coagulation is affected by...

Embodiment 2

[0042] Example 2 Exploration of the optimal density of the mononuclear cell separation solution of the striped Bamboo shark

[0043] In order to determine the optimal density of the mononuclear cell separation solution suitable for the striped bamboo shark, firstly, the human peripheral blood mononuclear cell separation solution was serially diluted with a large span using the shark diluent to prepare the striped bamboo shark mononuclear cell separation solution. After finding a suitable range, repeat the gradient dilution with a smaller span, and observe which dilution ratio range of the striped bamboo shark mononuclear cell separation solution has the best separation effect.

[0044] (1) Take commercially available human peripheral blood mononuclear cell separation liquid reagent (1.077g / mL) and use shark diluent to carry out a large-span gradient dilution to prepare the striped bamboo shark mononuclear cell separation liquid with different dilution ratios, A total of 8 grou...

Embodiment 3

[0051] Example 3 Detection of cell viability and purity in mononuclear cell layer after separation

[0052] In order to determine the purity of mononuclear cells and the proportion of viable cells in the buffy coat separated in step 6 in Example 2, observation and statistics were performed using an optical microscope and a hemocytometer, and the number of viable cells was determined using the trypan blue exclusion method. .

[0053] (1) Absorb the buffy coat layer of the step 6B3 group in Example 2, dilute it 20 times, add 0.4% trypan blue staining solution to it, make the final trypan blue concentration 0.04%, and carry out staining for 3 min.

[0054] (2) Draw the stained cells on a hemocytometer, observe under a light microscope, count the number of mononuclear cells, total viable cells and total cells in each counting cell, and calculate the average value. Statistics are shown in Table 1.

[0055] (3) According to the formula:

[0056] (4) After calculation, the numbe...

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Abstract

The invention belongs to the technical field of cell separation, and particularly relates to a shark mononuclear cell separation method, a shark diluent and application of the shark diluent. The shark diluent comprises a PBS (Phosphate Buffer Solution) and urea, and the density of the shark diluent is 1.0028 g / mL to 1.0235 g / mL. The shark mononuclear cell separation method mainly comprises the following steps: acquiring peripheral blood cells or tissue cells of sharks; diluting blood cells or tissue cells with a shark diluent to prepare a cell suspension; the shark mononuclear cell separating medium is prepared by diluting shark diluent and is used for separating mononuclear cells in shark blood and tissues. According to the shark mononuclear cell separation method provided by the invention, the pollution of red blood cells or granulocytes can be effectively reduced, so that the obtained mononuclear cells have relatively high purity and living cell proportion, the cost can be saved, and the effect is stable.

Description

technical field [0001] The invention belongs to the technical field of cell separation, and in particular relates to a method for separating shark mononuclear cells, a shark diluent and uses thereof. Background technique [0002] Peripheral blood mononuclear cells (PBMCs) refer to cells with only a single nucleus in peripheral blood, including lymphocytes and monocytes. The striped bamboo shark is a small benthic shark widely distributed in the southeastern waters of China. Because of its docile temperament and easy feeding, it is an important experimental animal in the field of single domain antibody (Single domain antibody) research in recent years. The mononuclear cells derived from the striped bamboo shark are the necessary raw materials for studying the immune response process of sharks and shark-derived single-domain antibodies. [0003] In the existing technical system, density gradient centrifugation with ficoll as the main separation liquid is an important method t...

Claims

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Application Information

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IPC IPC(8): C12N5/0786C12N5/0781C12N5/0783
CPCC12N5/0645C12N5/0635C12N5/0636C12N2509/10C12N2500/60C12N2500/30C12N2500/05Y02A50/30
Inventor 毕允晨邓鹏辉闻建晴王淏
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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