Determination of H2O2 Generation by pHPA Assay
采用pHPA实验测定H2O2生成
发布: 2016年11月20日第6卷第22期 DOI: 10.21769/BioProtoc.2010 浏览次数: 9029
评审: Ivan ZanoniAchille BroggiPinchas Tsukerman
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The authors used this protocol in:
Mar 2016
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Abstract
The production of reactive oxygen species, including H2O2, is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H2O2 release from cells (Panus et al., 1993; Murthy et al., 2010; Larson-Casey et al., 2016; Larson-Casey et al., 2014; He et al., 2011). This assay takes advantage of H2O2-mediated oxidation of horseradish peroxidase (HRP) to Complex I, which, in turn, oxidizes p-hydroxyphenylacetic acid (pHPA) to a stable, fluorescent pHPA dimer (2,2'-dihydroxy-biphenyl-5,5’ diacetate [(pHPA)2]). The H2O2-dependent HRP-mediated oxidation of pHPA is a sensitive and specific assay for quantifying H2O2 release from cells. This assay can measure H2O2 release from whole cells, mitochondria, or the NADPH oxidase.
Background
H2O2 generation primarily results from dismutation of superoxide anion (O2-), which occurs at a rapid rate (105-106 M-1 s-1) non-enzymatically. Unlike O2-, H2O2 can traverse membranes easily, so it is able to oxidize multiple molecules. ROS can be toxic to cells by oxidizing proteins, lipids, and nucleic acids and are associated with many human diseases. This protocol allows for detection of H2O2 release from NADPH oxidase or mitochondria in various cell types.
Materials and Reagents
- NuncTM 96-well black bottom plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 137101 )
- Eppendorf tubes
- 15 and 50 ml conical tubes
- Glucose (Sigma-Aldrich, catalog number: G7528 )
- HEPES (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080 )
- Sodium bicarbonate (NaHCO3) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S233-500 )
- 4-hydroxyphenylacetic acid (pHPA) (Sigma-Aldrich, catalog number: H50004 )
- HRP (Sigma-Aldrich, catalog number: P8125 )
- Ca2+/Mg2+/phenol red-free HBSS (Thermo Fisher Scientific, GibcoTM, catalog number: 14175-095 )
- α-Ketoglutaric acid (Sigma-Aldrich, catalog number: K2000 )
- Hydrogen peroxide solution (H2O2) (30%, w/w) (Sigma-Aldrich, catalog number: H1009 )
- Tris
- EDTA
- Sucrose
- Protease inhibitor tablets (Sigma-Aldrich, catalog number: 11836170001 )
- Phosphatase inhibitors (EMD Millipore, catalog number: 524625 )
- Antimycin A (optional)
- MitoTempo (optional)
- pHPA buffer (see Recipes)
- 1 mM H2O2 working stock solution (see Recipes)
- Mitochondrial buffer (see Recipes)
Equipment
- Kontes pellet pestle motor
- Centrifuge
- Plate reader (Molecular Devices, model: M2 SpectraMax )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Larson-Casey, J. L. and Carter, A. B. (2016). Determination of H2O2 Generation by pHPA Assay. Bio-protocol 6(22): e2010. DOI: 10.21769/BioProtoc.2010.
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分类
免疫学 > 免疫细胞功能 > 综合
细胞生物学 > 基于细胞的分析方法
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