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Method for detecting superoxide in cells

A technology of superoxide and detection method, which is applied in the field of superoxide detection in cells

Pending Publication Date: 2020-07-24
SICHUAN BRANCH OF CHINA TOBACCO +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Method for detecting superoxide in cells
  • Method for detecting superoxide in cells
  • Method for detecting superoxide in cells

Examples

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example 1

[0032] In this example, intracellular H after exposure to smoke condensates (Kentucky 3R4F cigarettes) prepared under different smoking modes 2 o 2 The visual detection of is as follows:

[0033] 1. Reagents and materials

[0034] Kentucky reference cigarette 3R4F (University of Kentucky, USA); human oral epidermoid carcinoma KB cells (Cell Resource Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences). α-MEM medium, sterilized PBS (Gibco, USA); fetal bovine serum (Sijiqing, Beijing); trypsin, dimethyl sulfoxide (biologically pure, DMSO, Amresco, USA), superoxide free radical detection fluorescence probe (BES-So-AM) and hydrogen peroxide detection fluorescent probe (BES-H 2 o 2 -Ac) (Wako Corporation, Japan).

[0035] 2. Preparation of flue gas condensate under different suction modes

[0036] Cigarette samples were extracted according to GB / T 5606.1-2004, and smoked with RM20H rotary table smoking machine under the two smoking conditions of ISO...

example 2

[0049] In this example, the visual detection of intracellular peroxide ion free radicals after exposure to smoke condensate (Kentucky 3R4F cigarettes) prepared under different smoking modes is as follows:

[0050] 1. Reagents and materials

[0051] Kentucky reference cigarette 3R4F (University of Kentucky, USA); human oral epidermoid carcinoma KB cells (Cell Resource Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences). α-MEM medium, sterilized PBS (Gibco, USA); fetal bovine serum (Sijiqing, Beijing); trypsin, dimethyl sulfoxide (biologically pure, DMSO, Amresco, USA), BES-So-AM and BES-H 2 o 2 -Ac (Wako Corporation, Japan).

[0052] 2. Preparation of flue gas condensate under different suction modes

[0053] Take cigarette samples according to GB / T 5606.1-2004, and use the RM20H rotary table smoking machine to smoke under the two smoking conditions of ISO and HCI respectively. According to the mass of TPM, a corresponding volume of DMSO was ad...

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Abstract

The invention discloses a method for detecting superoxide in cells. The method comprises the following steps of: dissolving a fluorescent probe by using dimethyl sulfoxide to serve as mother liquor, and preparing a cell culture medium into a cell culture solution containing the fluorescent probe by using a cell culture medium; culturing the cells in the cell culture medium containing fetal calf serum; extracting a cigarette sample, sucking, collecting total particulate matters of smoke, and adding dimethyl sulfoxide with corresponding volume according to the mass of the total particulate matters of the smoke to obtain a stock solution of the total particulate matters of the smoke; culturing the cells; and observing a fluorescent signal of the fluorescent probe in the cells by using a fluorescence microscope, and acquiring the concentration level of the superoxide in the cells. According to the method disclosed by the invention, the concentration level of active oxygen in the cells after the smoke is exposed is researched and used for evaluating the influence of smoke exposure on oxidative stress reaction of the cells, biological evaluation is performed on the safety of smoke components, and scientific reference is reversely provided for research and development and production of safe cigarettes.

Description

technical field [0001] The invention belongs to the technical field of biochemical analysis of intracellular active oxygen, and relates to a detection method of intracellular superoxide. Background technique [0002] Reactive oxygen species (Reactive oxygen species, ROS) is a class of small molecular groups with high chemical activity, including hydroxyl radicals, nitroxide radicals, alkoxyl radicals and peroxides, etc. Excessive ROS in cells Levels can cause damage to cell membranes, functional proteins, and nucleic acids, and may further lead to the occurrence of diseases. Cigarette smoke is an important pollutant in the living environment. It has been proved that it contains a large amount of active oxygen, mainly including alkoxy radicals and nitrogen oxide radicals in the gas phase, aromatic hydrocarbon radicals and quinone radicals in the particle phase, etc. , these active oxygen molecules entering the organism can affect the oxidative stress response in cells, and e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428G01N21/6402
Inventor 陶晓秋刘珊珊熊巍师默闻范子彦张海燕景浩王颖韶济民靳冬梅
Owner SICHUAN BRANCH OF CHINA TOBACCO