Hydrogen Peroxide Colorimetric Detection Kit

1. Use plate layout template to aid in proper sample and standard identification., 2. Pipet 50 µL SSA-treated samples, standards, or control into plate Wells., 3. Pipet 50 µL Sample Diluent into zero Wells., 4. Add 25 µL Fluorescent GSH Detection Substrate to each Well using a repeater pipet., 5. Gently tap plate sides to ensure adequate mixing of the reagents., 6. Incubate 15 minutes at room temperature., 7. Read the fluorescent emission at 510 nm with excitation at 370-410 nm. This data will be used to determine Free GSH concentration., 8. Add 25 µL Reaction Mixture to each Well using a repeater pipet., 9. Gently tap plate sides to ensure adequate mixing of the reagents., 10. Incubate 15 minutes at room temperature., 11. Read the fluorescence with excitation at 370-410 nm and emission at 510 nm. This data will be used to determine Total GSH concentration.

2 Clear Half-Area 96-Well MicroWell Plates, #267, Hydrogen Peroxide Standard (Hydrogen Peroxide at 1,000 µM in a special stabilizing solution), 200 µL, #6604, 5X Assay Buffer Concentrate (a buffer concentrate containing detergents and stabilizers), 25 mL, #6605, Colorimetric H2O2 Detection Substrate (a solution of the substrate in a special stabilizing buffer), 5 mL, #6608, 50X Horseradish Peroxidase Concentrate (a concentrated solution of HRP in a special stabilizing solution), 120 µL, #6609, Kit Manual

In biological systems incomplete reduction of O2 during respiration produces superoxide anion (O2-·), which is spontaneously or enzymatically dismutated by superoxide dismutase to H2O2. Many cells produce low levels of O2-· and H2O2 in response to a variety of extracellular stimuli, such as cytokines (TGF-ß1, TNF-a, and various interleukins), peptide growth factors (PDGF, EGF, VEGF, bFGF, and insulin), the agonists of heterotrimeric G protein–coupled receptors (GPCR) such as angiotensin II, thrombin, lysophosphatidic acid, sphingosine 1-phosphate, histamine, and bradykinin, and by shear stress. The addition of exogenous H2O2 or the intracellular production in response to receptor stimulation affects the function of various proteins, including protein kinases, protein phosphatases, transcription factors, phospholipases, ion channels, and G proteins. In 1894, Fenton described the oxidation of tartaric acid by Fe2+ and H2O2. H2O2 and O2 may participate in the production of singlet oxygen and peroxynitrite and the generation of these species may be concurrent with reactions involving iron, and under some circumstances they might be important contributors to H2O2 toxicity. A substantial portion of H2O2 lethality involves DNA damage by oxidants generated from iron-mediated Fenton reactions. Damage by Fenton oxidants occurs at the DNA bases or at the sugar residues. Sugar damage is initiated by hydrogen abstraction from one of the deoxyribose carbons, and the predominant consequence is eventual strand breakage and base release. ICT’s Hydrogen Peroxide Colorimetric Detection Kit is designed to quantitatively measure H2O2 in a variety of samples. This kit is validated for use in fresh urine, buffers, and tissue culture media (TCM). It is species independent. Please read the complete kit insert before performing this assay. A hydrogen peroxide standard is provided to generate a standard curve for the assay. All samples should be read off the standard curve. Samples are mixed with the Colorimetric H2O2 Detection Substrate and the reaction is initiated by addition of horseradish peroxidase (HRP). The reaction is incubated at room temperature for 15 minutes. The HRP reacts with the substrate in the presence of hydrogen peroxide to convert the colorless substrate into a colored product. The pink product is read at 560 nm. Increasing levels of H2O2 cause a linear increase in color. This kit is for research use only and is not for use in diagnostic procedures.