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Low-concentration ROS detection method and application thereof

A detection method and low-concentration technology, applied in measuring devices, using chemical indicators for analysis, instruments, etc., can solve problems such as inability to penetrate cell membranes, decrease in probe content, etc., to achieve increased fluorescence intensity, enhanced fluorescence intensity, and detection numerically significant effect

Active Publication Date: 2021-02-05
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that DCFH-DA can be hydrolyzed to DCFH under alkaline conditions, but DCFH cannot penetrate the cell membrane, resulting in a decrease in the content of the probe

Method used

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  • Low-concentration ROS detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 stimulates HeLa cells to generate ROS

[0046] (1) Use H 2 o 2 Stimulation of ROS production in HeLa cells

[0047] ①Use a pipette gun to absorb 89mL DMEM high-glucose medium, 10mL fetal bovine serum, and 1mL penicillin-streptomycin double antibody solution to prepare cell growth medium, and adjust the pH to 0.1M NaOH solution or 12M HCl solution by dropping 7.0.

[0048] ②The human cervical cancer cell line HeLa was selected as the cell line used in the experiment, and the cells were inoculated into a 96-well plate one day before the experiment, with 15,000 cells per well. The cells were grown on the wall for 12 hours until the confluence of the cells reached 50-70%.

[0049] ③ Add different volumes of H 2 o 2 Stimulate cells (make H 2 o 2 The concentrations in the system are respectively 0.03%, 0.06%), and cultured in the dark for 30 minutes in a cell culture incubator at 37°C.

[0050] ④Remove the solution after 30 min of stimulation, wash the cell...

Embodiment 2

[0056] Embodiment 2 detects H 2 o 2 Stimulated ROS in HeLa cells

[0057] ①The DCFH-DA solution (final concentration is 10 μM) diluted in high-glucose DMEM serum-free medium (containing 1% penicillin-streptomycin double antibody solution) was added to the oxidatively damaged HeLa cells in step (1) of Example 1, and passed Add 0.1M NaOH solution or 12M HCl solution dropwise to adjust the pH to 7.0, and incubate in the dark for 30 minutes.

[0058] ②Remove the solution after incubation for 30 minutes, and wash with 1×PBS (0.01M, pH=7.4) buffer solution for 2 to 3 times to fully remove the DCFH-DA solution that has not entered the cells, and obtain the sample to be tested.

[0059] ③ The sample to be tested is irradiated with LED light, the illumination time is 5min and 10min respectively, and the optical power density is 13.40mW / cm 2 .

[0060] ④ Use a multifunctional microplate reader to detect the fluorescence signal value in each well plate, wherein the excitation wavelen...

Embodiment 3

[0062] Example 3 Detection of ROS in HeLa cells stimulated by polystyrene plastic particles

[0063] ①The DCFH-DA solution (final concentration is 10 μM) diluted in high-glucose DMEM serum-free medium (containing 1% penicillin-streptomycin double antibody solution) was added to the oxidatively damaged HeLa cells in step (2) of Example 1, and passed Add 0.1M NaOH solution or 12M HCl solution dropwise to adjust the pH to 7.0, and incubate in the dark for 30 minutes.

[0064] ② After incubation for 30 minutes, remove the solution and wash with 1×PBS (0.01M, pH=7.4) buffer solution for 2-3 times to fully remove the DCFH-DA solution that did not enter the cells.

[0065] ③The solution to be tested is irradiated with LED light, the illumination time is 5min and 10min respectively, and the optical power density is 13.40mW / cm 2 .

[0066] ④ Use a multi-functional microplate reader to detect the fluorescent signal value in each well plate, wherein the excitation wavelength of the flu...

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Abstract

The invention discloses a detection method of low-concentration ROS and application thereof. The detection method disclosed by the invention is used for detecting the ROS level of reactive oxygen species in cells subjected to oxidative damage by utilizing a fluorescent probe DCFH-DA, and the method comprises the following steps: (1), adding a DCFH-DA solution into cells subjected to oxidative damage, performing incubating in a dark place, removing the solution, and washing the cells to obtain a cell sample to be detected; and (2), irradiating the to-be-detected cell sample with light, and detecting a fluorescence signal value. The detection method disclosed by the invention can be used for detecting low-dose ROS in cells, and the problems that the detection of the low-dose ROS is easily influenced by optical radiation and the real ROS level cannot be accurately expressed are solved; by adopting the method disclosed by the invention, the fluorescence intensity of different samples can be increased, the relative fluorescence intensity cannot be changed, and the equal-ratio increase of the fluorescence intensity of different samples is achieved, so that the detection numerical value is more obvious.

Description

technical field [0001] The invention relates to the technical field of biological activity detection, in particular to a low-concentration ROS detection method and its application. Background technique [0002] Reactive oxygen species (ROS) refer to oxygen-containing free radicals related to oxygen metabolism in organisms and highly active by-products in the form of non-free radicals, including superoxide anion, hydroxyl radical, hydrogen peroxide, etc. ROS participates in the body's immune response, gene expression regulation, and signal transduction in vivo, so its detection is of great significance in many scientific researches, such as oxidative stress characterization, pollutant toxicity research, and anti-tumor drug development. Excessive ROS will cause oxidative damage to the plasma membrane, protein, nucleic acid and other biomacromolecules, and the mechanism of oxidative damage is an important mechanism of toxicity of various pollutants. Therefore, it is important ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486G01N31/22
Inventor 侯森俞大良查英英
Owner JINAN UNIVERSITY
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