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Method for detecting content of active oxygen in cells

A technology of active oxygen content and detection method, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, measurement devices, etc.

Inactive Publication Date: 2016-12-07
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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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 content of active oxygen in cells
  • Method for detecting content of active oxygen in cells
  • Method for detecting content of active oxygen in cells

Examples

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Embodiment 1

[0025] A method for detecting intracellular active oxygen content, 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 species (ROS) in cells induced by ...

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Abstract

The invention discloses a method for detecting content of active oxygen in cells. The method has the advantage that a DCF (dichlorofluorescein) fluorescence signal value of a single live cell corresponding to pollutants exposed under different concentrations can be obtained; a detection result is accurate, and the problem of inaccurate measuring of final fluorescence intensity due to change of cell number caused by exposing of the pollutants is solved; the corresponding fluorescence signal value of single cell under a certain concentration can be truly reflected, and the injury to cells by pollutant exposing can be truly and accurately reflected, so as to lay a solid foundation for the application of an in-vitro cell model into the toxicity evaluation of the pollutants.

Description

technical field [0001] The invention relates to a method for detecting active oxygen content in cells, 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 the body. The traditional intracellu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N21/64
CPCG01N21/6402G01N21/6428G01N21/6452G01N21/6486G01N33/5014G01N33/5067G01N2021/6439
Inventor 吴兵刘苏张徐祥
Owner NANJING UNIV
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