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Hydrogen Peroxide Detection

Hydrogen Peroxide Detection by ADHP and Its Analogs



H2O2 dose responses were measured in a 384-well black solid plate with Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Cat# 11501). As low as 0.03 µM H2O2 was detected with 30 minutes incubation (n=3).
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.


H2O2 dose responses were measured in a 96-well black solid plate with Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Cat# 11502). As low as 0.03 µM H2O2 was detected.
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).
 

Table 1. Hydrogen Peroxide Assay Kits

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
11502Amplite® Fluorimetric Hydrogen Peroxide Assay Kit *Near Infrared Fluorescence*647670500 Tests
11501Amplite® Fluorimetric Hydrogen Peroxide Assay Kit *Red Fluorescence*571585500 Tests

 

Intracellular Hydrogen Peroxide Detection


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.


Images of live CHO-K1 cells in a 96-well plate. Live CHO-K1 cells were stained with Amplite® Intracellular Fluorimetric Hydrogen Peroxide Assay Kit (Cat# 11503). Left: Control cells. Right: Cells treated with 100 µM H2O2 at room temperature for 5 minutes.



The chemical structure of Dihydrofluorescein Diacetate (Cat# 15203).
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.
 

Table 2. Intracellular Hydrogen Peroxide Detection Assay and Probes

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
11503Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence*492515200 Tests
15204DCFH-DA [2',7'-Dichlorodihydrofluorescein diacetate] *CAS 4091-99-0*50452925 mg
15203Dihydrofluorescein diacetate [Fluorescin diacetate] *CAS 35340-49-9*49051425 mg
15206Dihydrorhodamine 123 *CAS 109244-58-8*50752910 mg
15207Dihydrorhodamine 123 *CAS 109244-58-8*50752910 mg