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Extraction method of mouse hepatic sinus endothelial cells

An endothelial cell and extraction method technology is applied in the field of extraction of mouse liver sinusoidal endothelial cells, which can solve the problems of insufficient purity and poor cell viability, and achieve the effects of high cell purity, low cost, and promoting apoptosis.

Inactive Publication Date: 2017-03-22
QINGDAO JIULONG BIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to make up for the defects of the prior art, which are easy to produce mechanical damage when separating mouse liver sinusoidal endothelial cells, poor cell viability, state decline, and insufficient purity caused by chemical pretreatment, and provide a method that can better maintain cell integrity. and biochemical activity, and the number of hepatic sinusoidal endothelial cells extracted at one time is large, and the purity is high. Extraction method of mouse hepatic sinusoidal endothelial cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0056] Experimental example 1: Microscopic observation

[0057] Observe the obtained mouse liver sinusoidal endothelial cells under an inverted microscope. Under low power, living cells can be seen to be bright round, full, with good light transmittance, and clear nuclei. Approximately 2×106 sinusoids can be obtained per mouse. Endothelial cells.

experiment example 2

[0058] Experimental example 2: Scanning electron microscope (SEM) observation

[0059] The hepatic sinusoidal endothelial cells obtained in Example 1 were inoculated on a cell slide pre-plated with rat tail collagen type I, fixed overnight at 4°C with glutaraldehyde, dehydrated with a gradient of 30% to 100% alcohol, with a gradient of 10%, each gradient About 2h, after dehydration, vacuum drying and spraying gold on environmental scanning electron microscopy, it can be seen that the cells are spindle-shaped, with large nuclei, thin surroundings, and window structures of different sizes can be seen.

experiment example 3

[0060] Experimental example 3: Immunofluorescence (IF) identification

[0061] 1) Add type I rat tail collagen to the cell climbing sheet and dry it for later use;

[0062] 2) Inoculate hepatic sinusoidal endothelial cells (LSEC) on the cell slide;

[0063] 3) Wash the cells with PBS for 5min×3 times;

[0064] 4) Fixed with 4% paraformaldehyde at 4℃ for 1 hour;

[0065] 5) Wash with PBS 5min×1 times;

[0066] 6) 0.05% Triton-100 breaks the membrane at 4°C for 10 minutes;

[0067] 7) 5% BSA sealed at 4℃ for 1 hour;

[0068] 8) cd31 antibody 1:50, overnight at 4°C in a humid box;

[0069] 9) Wash with PBS for 5min×3 times;

[0070] 10) Incubate the FITC-labeled fluorescent secondary antibody in a humid box at 4°C for 1 hour;

[0071] 11) DAPI 20ul drops on the surface of the slide to stain the nucleus for 10 minutes;

[0072] 12) Wash with PBS for 5min×3 times;

[0073] 13) Mount the slides with glycerin mounting tablets, and take photos with a fluorescence microscope.

[0074] The cells are spind...

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Abstract

The invention discloses an extraction method of mouse hepatic sinus endothelial cells. According to the method, Kupffer cells are removed through gadolinium chloride injection, and separation of the hepatic sinus endothelial cells is achieved by combining two-step perfusion, differential centrifugation, percoll centrifugation and differential attachment. The extraction method of the mouse hepatic sinus endothelial cells is simple, easy to operate and high in cell purity and harvest rate, complete structural and functional activity is kept in in-vitro culture, and therefore the method can be applied in biochemistry and immunology. In addition, laboratorial conventional instruments are used in the method, the cost is low, and therefore the method can be popularized in most laboratories.

Description

Technical field [0001] The invention relates to a method for extracting cells, in particular to a method for extracting mouse liver sinusoidal endothelial cells. Background technique [0002] Liver Sinusoidal Endothelial Cells (Liver Sinusoidal Endothelial Cells) are non-substantial cells in the liver, which play a role in barrier, material metabolism, ultrafiltration, endocytosis, and antigen presentation in the liver. In recent years, domestic and foreign scholars have conducted a lot of research on the isolation method of hepatic sinusoidal endothelial cells, but it is still difficult to obtain hepatic sinusoidal endothelial cells with high purity, good cell viability and stable function, especially mouse hepatic sinusoidal endothelial cells. Due to the rich strains of mice, which can more fully meet the needs of various researches, it is of great significance to obtain mouse hepatic sinusoidal endothelial cells with good cell viability. [0003] Currently, mouse hepatic sinuso...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/067C12N2509/00
Inventor 刘乃山宋延超迟培升
Owner QINGDAO JIULONG BIO PHARMA
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