Hydrogen peroxide assay

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1. Hydrogen peroxide assay

Hydrogen peroxide assay를 통해 세포 내 ROS의 양을 정량한다. DCF-DA는 세포 안으로 확산되어 esterase에 의해 비형광물질이 된 후 ROS와 반응하여 형광성을 띠는 DCF가 된다. 따라서 DCF 형광도가 높으면 세포 내 ROS 활성이 높음을 의미한다. PBS wash하는 이유는 세포 외 ROS와 반응을 배제하기 위함이다. 실험결과 형광도는 A>C>B 순서대로 크며 편차를 고려하여도 이 관계가 역전되지 않는다. 화합물이 항산화 효과가 있다면 형광도가 가장 큰 A가 H₂O₂ treated, 가장 작은 B가 nontreated, 그 중간의 값을 갖는 C가 H₂O₂ and chemical treated일 것이다. 항산화 chemical 처리로 인해 ROS 활성이 감소하여 H₂O₂ 단독처리한 A보다 chemical 첨가한 C의 형광도가 낮은 것이다.
2. Dihydroethidium (DHE) assay

Dihydroethidium(DHE) assay는 생체 내 ROS 양을 측정하는 실험이다. 원리는 DHE가 superoxide(O₂ )와 산화반응으로 2-hydroxyethidium(2-OH-E , ex 500-530 nm/em 590-620 nm)을 형성하거나 다른 ROS와 비특이적 산화반응으로 ethidium(E , ex 480 nm/em 576 nm)을 형성하는 것이다.

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1. Hydrogen peroxide assay

The hydrogen peroxide (H2O2) assay is a widely used analytical technique for the detection and quantification of hydrogen peroxide, a reactive oxygen species (ROS) that plays a crucial role in various biological processes. This assay is particularly valuable in the field of oxidative stress research, as it allows researchers to measure the levels of H2O2 in biological samples, providing insights into the cellular redox status and potential oxidative damage. The hydrogen peroxide assay typically involves the use of a chromogenic or fluorogenic reagent that reacts with H2O2 to produce a measurable signal, such as a color change or fluorescence. Common reagents used in this assay include horseradish peroxidase (HRP) coupled with a substrate like Amplex Red or 2',7'-dichlorofluorescin diacetate (DCFDA). The intensity of the signal generated is proportional to the concentration of H2O2 in the sample, allowing for quantitative analysis. One of the key advantages of the hydrogen peroxide assay is its sensitivity and specificity. With proper sample preparation and assay optimization, researchers can detect H2O2 at low micromolar or even nanomolar concentrations, making it a valuable tool for studying oxidative stress in various biological systems, such as cell cultures, tissue samples, or even in vivo models. Additionally, the assay can be adapted to different experimental setups, including plate-based formats for high-throughput screening or more specialized techniques like flow cytometry or microscopy-based imaging. However, it is important to note that the hydrogen peroxide assay is not without its limitations. Factors such as sample matrix, the presence of interfering compounds, and the potential for cross-reactivity with other ROS can affect the accuracy and reliability of the results. Careful experimental design, appropriate controls, and thorough data analysis are crucial to ensure the validity of the findings. Overall, the hydrogen peroxide assay remains an essential tool in the field of oxidative stress research, providing researchers with a reliable and sensitive method to quantify H2O2 levels and gain insights into the complex interplay between oxidative stress and various biological processes.
2. Dihydroethidium (DHE) assay

The dihydroethidium (DHE) assay is a widely used technique for the detection and quantification of superoxide (O2•-), a reactive oxygen species (ROS) that plays a crucial role in various physiological and pathological processes. This assay is particularly valuable in the field of oxidative stress research, as it allows researchers to measure the levels of superoxide in biological samples, providing insights into the cellular redox status and potential oxidative damage. The DHE assay relies on the oxidation of the non-fluorescent dihydroethidium (also known as hydroethidine) to the fluorescent product ethidium, which intercalates with DNA and emits a red fluorescence. The intensity of the fluorescence signal is proportional to the concentration of superoxide in the sample, allowing for quantitative analysis. One of the key advantages of the DHE assay is its specificity for superoxide. Unlike some other ROS detection methods, the DHE assay is relatively selective for superoxide, making it a valuable tool for studying the role of this particular ROS in various biological processes. Additionally, the assay can be adapted to different experimental setups, including plate-based formats for high-throughput screening or more specialized techniques like flow cytometry or microscopy-based imaging. However, it is important to note that the DHE assay is not without its limitations. Factors such as sample matrix, the presence of interfering compounds, and the potential for non-specific oxidation of DHE can affect the accuracy and reliability of the results. Careful experimental design, appropriate controls, and thorough data analysis are crucial to ensure the validity of the findings. Furthermore, the DHE assay is not without its challenges in terms of interpretation. The conversion of DHE to ethidium can be influenced by various factors, including the presence of other ROS, enzymatic activities, and cellular redox status. Researchers must exercise caution when interpreting the results and consider the potential confounding factors that may influence the observed fluorescence signal. Despite these limitations, the DHE assay remains an essential tool in the field of oxidative stress research, providing researchers with a reliable and specific method to quantify superoxide levels and gain insights into the complex interplay between oxidative stress and various biological processes.

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