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Method of using Hoechst 33258 fluorescent dye to detect content of active oxygen in living cells

A technology of active oxygen content and fluorescent dyes, applied in fluorescence/phosphorescence, material analysis through optical means, measuring devices, etc., can solve problems such as inaccurate detection results, and achieve accurate, true and accurate reflection of detection results

Inactive Publication Date: 2016-12-07
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 of using Hoechst 33258 fluorescent dye to detect content of active oxygen in living cells
  • Method of using Hoechst 33258 fluorescent dye to detect content of active oxygen in living cells
  • Method of using Hoechst 33258 fluorescent dye to detect content of active oxygen in living cells

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Embodiment 1

[0025] A method for detecting active oxygen content in living cells by using Hoechst 33258 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 ...

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Abstract

The invention discloses a method of using a Hoechst 33258 fluorescent dye to detect the content of active oxygen in living cells. The method can obtain the DCF fluorescent signal values of a single living cell corresponding to exposure pollutants with different concentrations. The detection result of the method is precise. The problem that the final fluorescent strength measurement is not accurate due to the cell number change caused by pollutant exposure is solved. The provided detection method can faithfully represent the fluorescent value of a single cell corresponding to a certain concentration. Thus the damage, which is caused by pollutant exposure, to the cells can be faithfully and accurately represented. The method provides a more solid foundation for the application of an in-vitro cell model in the pollutant toxicity evaluation.

Description

technical field [0001] The invention relates to a method for detecting active oxygen content in living cells by using Hoechst 33258 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 d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 吴兵刘苏张徐祥
Owner NANJING UNIV
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