Extracellular H2O2 Exposure Model System
Exposure to hydrogen peroxide can inhibit cell proliferation, cause oxidative damage of macromolecules in cells, and eventually lead to serious consequences such as cell aging, death and mutation. The Fenton reaction between hydrogen peroxide and Fe2+ ions produces highly active OH radicals, which is considered to be the main mechanism of oxidative damage. Therefore, H2O2 induced oxidative stress cell model is widely used to explore the mechanism of free radical mediated cell damage and the protection and repair mechanism of antioxidants on oxidative damage
To help our customers to study and culture various cell types under specific oxidative stress conditions can build a cell oxidative stress model. Creative Bioarray has established a set of workflow of inducing cell oxidative stress with hydrogen peroxide, which can establish various types of oxidative stress models for customers.
Induction of Cellular Oxidative Stress
Fig. 1 Extracellular H2O2 Exposure Model System (Sies, 2017)
In order to expose cells for a longer time, we use glucose oxidase to catalyze the oxidation of glucose to gluconic acid to produce H2O2. In this way, a continuous H2O2 flux can be generated over a longer period of time. We will monitor the actual H2O2 concentration in the medium, which is very important for accurately measuring the cumulative dose of the application, because the extracellular concentration will be the result of H2O2 produced by enzymatic reaction and H2O2 decomposed by cells.
Dose Response of H2O2 Toxicity
In order to determine the dose toxicity of H2O2 to cells, the survival rate was evaluated after 24 hours of exposure to different concentrations of H2O2 by MTT method to determine the appropriate induction dose.
SOP for Cell Stress Model Evaluation
We will evaluate the oxidative stress of cells through the following parameters
- Loss of clonal cell survival, inhibition of DNA synthesis, decrease in NAD levels, decrease in ATP levels, DNA strand breaks, chromosomal aberrations, sister-chromatid exchanges, mutations.
- The growth changes of cells treated with H2O2 were indirectly measured by measuring the absorbance of MTT dye.
- The intracellular ROS level was detected by oxidation sensitive fluorescent probe dye dihydroethidium.
- Intracellular glutathione levels were analyzed using 5-chloromethylfluorescein diacetate.
- The activities of SOD and catalase were measured.
Cell Oxidative Model Construction Service Justification
Constructing various cell models of oxidative stress is the basis of cell aging, antioxidants and so on. The in vitro test system using cultured cells will be a useful intermediate screening test, which can avoid unnecessary animal experiments. Generally speaking, it is necessary to evaluate the substance with a simple in vitro test system, and then confirm the results with in vitro and in vivo test systems. In order to detect the antioxidant effect of a given substance in an in vitro system, excess oxidants are usually injected into a cell model to obtain rapid results.
Creative Bioarray is dedicated to providing high-quality products, comprehensive services, and tailored solutions to support and facilitate life sciences and pharmaceutical research and development. If you have any questions or needs, please contact us, and our customer service staff will help you at the first time.
Reference
- Sies, H. (2017). Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress. Redox Biology, 11, 613-619.
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