Cell DNA damage detection method

A technology for DNA damage and detection methods, which is applied in the direction of measuring devices, instruments, and material analysis through electromagnetic means, can solve the problems of sophisticated and complex production processes, high hardware and technical requirements, and achieve simplified preparation processes and improved efficiency , the effect of not easy cell overlap

Active Publication Date: 2014-04-30
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The microchamber array method improves the preparation process of the glue plate compared with the conventional SCGE, and the position of each cell in the microchamber is clear, and automatic analysis is used to ensure that every cell is analyzed. However, the microchamber mold manufacturing process in the microchamber array method

Method used

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  • Cell DNA damage detection method
  • Cell DNA damage detection method
  • Cell DNA damage detection method

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0034] Example 1:

[0035] (1) Preparation of mouse (Cavia porcellus) spleen cell sample: The mouse was killed by cervical dislocation. After a simple treatment with 75% ethanol, the spleen was quickly taken out in an ultra-clean table, placed in a glass dish, and rinsed with pre-cooled PBS buffer After the blood on the surface, soak it in PBS buffer, and cut it into 1~2mm with ophthalmic surgical scissors 3 Of small pieces. The PBS buffer with small pieces of spleen suspended is sucked into a disposable sterile syringe (with the needle removed) and repeatedly filtered through a 300-mesh cell sieve to collect the cell suspension. Centrifuge at 1500 rpm for 10 minutes at room temperature to obtain cell pellet, then resuspend it in RMPI-1640, and adjust the cell density to about 8×10 5 Pieces / mL.

[0036] (2) Cell pollution: Inoculate the cells in a 24-well cell culture plate, and then add H 2 O 2 Solution, H 2 O 2 The concentrations were 1μmol / L, 20μmol / L, and 40μmol / L, and a blank...

Example Embodiment

[0045] Example 2:

[0046] (1) Preparation of human acute monocytes (THP-1) samples: THP-1 cells were cultured in RMPI1640 medium containing 10% fetal bovine serum, 100U / mL penicillin, and 100U / mL streptomycin at 37°C, 5 %CO2 cell culture incubator. When passaging, use a pipette to gently pipette the cells evenly and then pass them directly. According to the needs of the experiment, the cells are seeded in a 24-well cell culture plate and cultured for 24 hours for later use.

[0047] (2) Cell pollution: inoculate the cells in a 24-well cell culture plate, and then add HgCl 2 Solution, HgCl 2 The concentrations were 1μmol / L, 10μmol / L, 100μmol / L, and a blank control group was set up, and the exposure was carried out at 4℃ for 1.5h. After the flooding is over, wash twice with PBS, collect the cells by centrifugation at 1200 rpm, and resuspend them in PBS to adjust the cell density to about 8×10 5 Pieces / mL to be used.

[0048] (3) Follow-up operations refer to steps 3 to 9 in Example ...

Example Embodiment

[0049] Example 3:

[0050] (1) Preparation of mouse (Cavia porcellus) spleen cell samples refer to step 1 of Example 1.

[0051] (2) Cell pollution: inoculate the cells in a 24-well cell culture plate, and then add CuCl 2 Solution, CuCl 2 The concentrations were 10nmol / L, 100nmol / L, 1μmol / L, and a blank control group was set up, and the exposure was carried out at 4℃ for 1.5h. After the flooding is over, wash twice with PBS, collect the cells by centrifugation at 1000 rpm, and resuspend them in PBS to adjust the cell density to about 8×10 5 Pieces / mL to be used.

[0052] (3) Follow-up operations refer to steps 3 to 9 in Example 1. See the results of the experiment image 3 .

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Abstract

The invention discloses a cell DNA damage detection method, and relates to the cell damage detection. The cell DNA damage detection method comprises the following steps: preheating a to-be-detected cell sample with low-gelling temperature agarose prepared from calcium and magnesium-free PBS (Phosphate Buffer Solution) to obtain a mixed liquid, paving the mixed liquid in a plate cover sample hole of a 24-hole cell culturing plate, spreading agarose evenly, cooling, curing, and then immersing the plate cover into cell lysis buffer, and pyrolyzing; washing an agarose plate after pyrolysis by an alkaline unwinding liquid, and then immersing the agarose plate into a container filled with a precooling alkaline unwinding liquid; placing the agarose plate after alkaline unwinding into an electrophoresis tank filled with a precooling electrophoresis liquid for electrophoresis; immersing the agarose plate after the electrophoresis into milli-Q water; standing and immersing the agarose plate by precooling absolute ethyl alcohol, and refrigerating and storing the taken agarose plate; adding a drop of MillI-Q water dropwise in each hole of the agarose plate after dehydration and drying, standing, wetting the agarose plate, adding 10-15micron L of ethidium bromide dropwise in each hole, keeping away from light, dyeing, sucking residual dye liquid dry on the surface of the agarose plate by filtration paper, and collecting images with excitation wavelength being 515nm.

Description

technical field [0001] The invention relates to cell damage detection, in particular to a cell DNA damage detection method. Background technique [0002] In cancer patients, the stability of DNA genetic information is damaged to varying degrees, which indicates that the occurrence of cancer is closely related to DNA damage. There are various harmful physical and chemical factors in the environment (such as ultraviolet radiation, pesticide residues and other organic pollutants, etc.) that can directly or indirectly cause cell DNA damage, and a large number of new factors enter the environment every year, so Rapid and effective detection of DNA damage and toxicity of existing and newly entered environmental factors is conducive to taking timely measures to control and treat harmful factors, reducing the occurrence of related cancers and genetic diseases, and is of great significance for environmental monitoring and protection. [0003] Single cell gel electrophoresis (Single ...

Claims

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Application Information

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IPC IPC(8): G01N21/63G01N27/447
Inventor 韩大雄肖丹王海燕
Owner XIAMEN UNIV
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