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An Application of the Amplite® Fluorimetric Hydrogen Peroxide Assay Kit
The Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Cat#11501) is an essential tool for studying the relationship between reactive oxygen(H2O2) species (ROS) activity and proper cellular function. Among ROS, hydrogen peroxide is a major regulator and by-product in many signal transduction pathways. Consequently, researchers will often use the kit to quantify intracellular H2O2 to gauge the oxidative state of their cellular subjects. This measurement has been applied to determine levels of inflammation, occurrence of apoptosis, and the degree of other signs of oxidative stress and imbalance.
In a recent study published in BioMed Research International (Nagata, Ito, Itoga, Kanazawa, & Masaki, 2015), the kit was used to evaluate the cytoxicity of phenolic compounds on melanocytes. Specifically, the study focused on rhododendrol (RD) and raspberry ketone (RK) which is a derivative of RD, as well as several other phenolic compounds. RD itself is best known as a skin-whitening agent commonly found in Asian cosmetic products. In past years there have been reports of RD inducing leukoderma, or the appearance of unseemly white spots on the skin, indicating the death of melanocytes. This phenomenon calls for more examination on the mechanisms involved in leukoderma, which leads to this study (Nagata et al., 2015) scrutinizing the effect of phenols on melanocytes by observing generated ROS, including H2O2.
The Amplite® Hydrogen Peroxide Kit played an essential role in acquiring quantitative measurements of intracellular H2O2 levels in B16F10 melanoma cells treated with RD or RK. The H2O2 values were then interpreted as the relative level of cytotoxic effect that RD and RK had on the B16F10 cells. The cytoxicity of RD and RK was found to be higher on the B16F10 cells than on the control HaCat keratinocytes, suggesting that the cytotoxic effect of RD is melanocyte-specific. This conclusion was attainable with the help of the Amplite® Hydrogen Peroxide Kit, which is robust enough to be applied to various studies investigating the role of H2O2 and other agents in progressing abnormal conditions such as leukoderma.
The signature dye used in is Amplite® Red, a peroxidase substrate that generates a resorufin product with a brilliant red hue upon reacting with traces of H2O2 or HRP. This product emits a signal that can be read either spectrophotometrically or fluorimetrically at Ex/Em= 571/585nm. It enables the user to detect H2O2 concentrations as sparse as 10 picomoles in a 100uL assay volume. As a simple "mix and read" procedure, the assay requires minimal prep time and can also be used directly on live cells. With its sensitivity, flexibility, and ease of use, it's reasonable to see why researchers have chosen to incorporate the Amplite® Hydrogen Peroxide Kit into their studies.
In a recent study published in BioMed Research International (Nagata, Ito, Itoga, Kanazawa, & Masaki, 2015), the kit was used to evaluate the cytoxicity of phenolic compounds on melanocytes. Specifically, the study focused on rhododendrol (RD) and raspberry ketone (RK) which is a derivative of RD, as well as several other phenolic compounds. RD itself is best known as a skin-whitening agent commonly found in Asian cosmetic products. In past years there have been reports of RD inducing leukoderma, or the appearance of unseemly white spots on the skin, indicating the death of melanocytes. This phenomenon calls for more examination on the mechanisms involved in leukoderma, which leads to this study (Nagata et al., 2015) scrutinizing the effect of phenols on melanocytes by observing generated ROS, including H2O2.
The Amplite® Hydrogen Peroxide Kit played an essential role in acquiring quantitative measurements of intracellular H2O2 levels in B16F10 melanoma cells treated with RD or RK. The H2O2 values were then interpreted as the relative level of cytotoxic effect that RD and RK had on the B16F10 cells. The cytoxicity of RD and RK was found to be higher on the B16F10 cells than on the control HaCat keratinocytes, suggesting that the cytotoxic effect of RD is melanocyte-specific. This conclusion was attainable with the help of the Amplite® Hydrogen Peroxide Kit, which is robust enough to be applied to various studies investigating the role of H2O2 and other agents in progressing abnormal conditions such as leukoderma.
The signature dye used in is Amplite® Red, a peroxidase substrate that generates a resorufin product with a brilliant red hue upon reacting with traces of H2O2 or HRP. This product emits a signal that can be read either spectrophotometrically or fluorimetrically at Ex/Em= 571/585nm. It enables the user to detect H2O2 concentrations as sparse as 10 picomoles in a 100uL assay volume. As a simple "mix and read" procedure, the assay requires minimal prep time and can also be used directly on live cells. With its sensitivity, flexibility, and ease of use, it's reasonable to see why researchers have chosen to incorporate the Amplite® Hydrogen Peroxide Kit into their studies.
References
- Takeshi Nagata, Shinobu Ito, Kazuyoshi Itoga, Hideko Kanazawa, and Hitoshi Masaki, The Mechanism of Melanocytes-Specific Cytotoxicity Induced by Phenol Compounds Having a Prooxidant Effect, relating to the Appearance of Leukoderma, BioMed Research International, vol. 2015, Article ID 479798, 12 pages, 2015. doi:10.1155/2015/479798
- Amplite® Fluorimetric Hydrogen Peroxide Assay Kit. AAT Bioquest, n.d. Web. 22 June 2016
Original created on November 27, 2019, last updated on November 27, 2019
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