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Method for detecting reactive oxygen content in living cells through DAPI fluorescent dye

A technology of active oxygen content and fluorescent dyes, which is applied in the direction of fluorescence/phosphorescence, preparation of test samples, material analysis through optical means, etc., can solve the problems of inaccurate detection results and achieve accurate detection results

Inactive Publication Date: 2016-11-09
NANJING UNIV
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Problems solved by technology

However, in the existing technology, the change of the final fluorescence intensity caused by the change of the cell number is often ignored, resulting in inaccurate detection results

Method used

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  • Method for detecting reactive oxygen content in living cells through DAPI fluorescent dye
  • Method for detecting reactive oxygen content in living cells through DAPI fluorescent dye
  • Method for detecting reactive oxygen content in living cells through DAPI fluorescent dye

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Embodiment 1

[0025] A method for detecting active oxygen content in living cells by using DAPI fluorescent dye, comprising the steps of:

[0026] Step 1: Select the human liver cancer cell line HepG2 as the cell line for the test, observe its growth state under a microscope, remove the original medium, wash the cells with PBS buffer 1 or 2 times, and add 1 mL of trypsin-EDTA to the culture dish Solution, digest the cells; after the cell digestion is complete, add 10mL of fresh medium containing serum to stop the digestion, and blow the obtained cell suspension evenly;

[0027] Step 2, measure the cell density in the cell suspension with a hemocytometer, and seed the cells into a 96-well plate at a density of 10,000 cells per well;

[0028] Step 3: 24 hours after seeding the plate, the cells were exposed to poison, and different concentrations of exposed pollutant arsenic were added to different well plates;

[0029] Step 4: Use the DCFH-DA probe to detect the content of reactive oxygen sp...

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Abstract

The invention discloses a method for detecting reactive oxygen content in living cells through DAPI fluorescent dye. According to the method, the DCF fluorescence signal values of the single living cells corresponding to exposed contaminants at different concentrations can be obtained. According to the method for detecting the reactive oxygen content in the living cells, the detecting result is precise, the problem that due to the fact that the contaminants are exposed, the number of the cells changes, and final fluorescence intensity measurement is inaccurate is solved, the fluorescence signal values of the single living cells corresponding to a certain concentration can be truly reflected through the method for detecting the reactive oxygen content, therefore, the damage of contaminant exposure to the cells can be truly and accurately reflected, and a more solid foundation is laid for application of an in-vitro cell model in contaminant toxicity assessment.

Description

technical field [0001] The invention relates to a method for detecting active oxygen content in living cells by using DAPI fluorescent dye, belonging to the field of bioactive cell detection. Background technique [0002] In vitro cell model is a technology that simulates the growth environment of organisms in vitro and detects the influence of external stimuli on cells. The use of cells as the test object has the advantages of high sensitivity, greatly shortening the test cycle, and easy to find out the mechanism of action. It not only improves the efficiency of toxicology testing but also reduces the use of animals. It is an important platform in the biological evaluation system. Oxidative damage mechanism is an important mechanism of toxicity for many pollutants. In the process of using in vitro cell models to evaluate the toxicity of pollutants, it is often necessary to detect the content of reactive oxygen species (ROS) in cells to characterize the oxidative damage to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/543G01N1/28
CPCG01N1/28G01N21/6486G01N33/543
Inventor 吴兵刘苏张徐祥
Owner NANJING UNIV
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