Hydrogen Peroxide Detection

Hydrogen Peroxide Detection by ADHP and Its Analogs

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Hydrogen peroxide (H₂O₂) is a reactive oxygen metabolic by-product that serves as a key regulator for a number of oxidative stress-related states. It is involved in a number of biological events that have been linked to asthma, atherosclerosis, diabetic vasculopathy, osteoporosis, a number of neurodegenerative diseases and Down's syndrome. Perhaps the most intriguing aspect of H₂O₂ biology is the recent report that antibodies have the capacity to convert molecular oxygen into hydrogen peroxide to contribute to the normal recognition and destruction processes of the immune system. Measurement of this reactive species will help to determine how oxidative stress modulates varied intracellular pathways.

Amplite^® Fluorimetric Hydrogen Peroxide Assay Kit (Cat# 11501) uses our non-fluorescent Amplite^® Red peroxidase substrate to quantify hydrogen peroxide in solutions and cell extracts. It can also be used to detect a variety of oxidase activities through enzyme-coupled reactions. The kit is an optimized "mix and read" assay that is compatible with HTS liquid handling instruments. It provides a sensitive, one-step fluorimetric assay to detect as low as 3 picomoles of H₂O₂ in a 100 µL assay volume (30 nM). The assay can be performed in a convenient 96-well or 384-well microtiter plate format and readily adapted to automation. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = ~540/590 nm or an absorbance microplate reader at ~570 nm.

Amplite^® Fluorimetric Hydrogen Peroxide Assay Kit (Cat# 11502) uses our unique Amplite^® IR peroxidase substrate to quantify hydrogen peroxide in solutions and cell extracts. Amplite^® IR generates the fluorescence that is pH-independent from pH 4 to 10. It is a superior alternative to ADHP (Amplex^® Red) for the detections that require low pH where ADHP has reduced fluorescence. In addition, Amplite^® IR generates a product that has maximum absorption at 647 nm with maximum emission at 670 nm. The near infrared fluorescence minimizes the assay background that is often caused by the autofluorescence of biological samples. The kit can also be used to detect a variety of oxidase activities through enzyme-coupled reactions. Amplite^® Fluorimetric Hydrogen Peroxide Assay Kit (Cat# 11502) provides a sensitive, one-step fluorimetric assay to detect as little as 3 picomoles of H₂O₂ in a 100 µL assay volume (30 nM).

Intracellular Hydrogen Peroxide Detection

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Amplite^® Intracellular Fluorimetric Hydrogen Peroxide Assay Kit (Cat# 11503) uses our unique ROS Green™ to quantify hydrogen peroxide in live cells. Cell-permeable ROS Green™ generates green fluorescence when it reacts with hydrogen peroxide. The kit is in an optimized "mix and read" assay format that is compatible with HTS liquid handling instruments. Amplite^® Intracellular Fluorimetric Hydrogen Peroxide Assay Kit provides a sensitive, one-step fluorimetric assay to detect H₂O₂ in live cells. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format. Its signal can be easily read by either a fluorescence microplate reader at Ex/Em = 490/520 nm for H₂O₂ detection or a fluorescence microscopy.



Dihydrofluorescein diacetate (also called fluorescein diacetate, Cat# 15203) is hydrolyzed by cellular esterases to dihydrofluorescein (also called fluorescein), which is oxidized to fluorescein primarily by H₂O₂. Dihydrofluorescein diacetate might be reactive toward a broad range of oxidizing reactions that may be increased during intracellular oxidant stress. Cell-loading studies indicated that dihydrofluorescein diacetate achieves higher intracellular concentrations than other redox sensors, such as 2',7'-dichlorodihydrofluorescein diacetate and dihydrorhodamine 123.

2',7'-Dichlorodihydrofluorescein diacetate (also called 2',7'-dichlorofluorescein diacetate, Cat# 15204) works similarly to dihydrofluorescein diacetate (Cat# 15203). 2',7'-Dichlorodihydrofluorescein diacetate has lower pKa, making this probe superior for the assays that require low pH.

Dihydrorhodamine 123 (DHR 123, Cat# 15206) is by far the most-used probe for the measurement of intracellular H₂O₂. DHR 123 is oxidized directly to rhodamine 123, which is excitable at 488 nm and emits at 515 nm in the same emission range as FITC. It is widely used in human neutrophils, human eosinophils, HL60 cells, rat mast cells, guinea pig neutrophils, cultured chondrocytes, rat brain, rat renal proximal tubular cells, mesangial cells and L929 cells. In combination with other fluorescent reagents (such as surface receptor analysis using fluorescent antibodies, cell viability using propidium iodide, and calcium indicators), this probe can be used for multiplex measurements.