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Reactive Oxygen Species (ROS)

MitoROS
Reactive Oxygen Species
Reactive oxygen species (ROS) are chemically reactive, oxygen-containing molecules that are naturally produced as a byproduct of cellular metabolism. Under physiological conditions, ROS levels are carefully regulated, where they operate as messengers in normal cell signal transduction, cell cycling, gene expression and homeostasis.

When generated in excess, ROS has the potential to cause a number of deleterious events. At high concentrations, ROS induces oxidative stress in cells resulting in subsequent damage of cellular macromolecules such as nucleic acids, membrane lipids and cellular proteins. Oxidative damage of these biomolecules can trigger cell apoptosis, is associated with aging, and has been implicated in the pathogenesis of a variety of diseases, cancers and neurodegenerative disorders, as well as, diabetes and inflammation.

Because of its damaging effects, cells have several carefully regulated systems for managing excess ROS. The most well studied system is the glutathione-ascorbate cycle, which detoxifies H2O2 into H2O, using NADH and NADPH as electron donors. Other systems include enzymes such as superoxide dismutase, which catalyzes the dismutation of the superoxide anion (O2-) into O2 or H2O2, and catalase, which catalyzes the decomposition of H2O2 into H2O and O2.

 

 

Types of ROS


Most cellular ROS are generated endogenously as byproducts of mitochondrial oxidative phosphorylation, or formed as intermediates of oxidoreductase enzymes and metal catalyzed oxidation. Since oxygen atoms contain two unpaired electrons in separate orbits of its outer electron shell, it is susceptible to radical formation. The sequential reduction of oxygen through the addition of electrons leads to the formation of a number of ROS including superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), hypochlorous acid (HOCl), peroxynitrite anion (ONOO-), and nitric oxide (NO).

ROS Flow Chart

Reduction of oxygen and its byproducts.

 

Hydrogen Peroxide


Hydrogen peroxide (H2O2) is a reactive oxygen metabolic byproduct 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 neurodegeneration 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.

Intracellular Hydrogen Peroxide Probes

DCFH-DADihydrorhodamine 123OxiVision™ BlueOxiVision™ Green
PrincipleEsterases cleave off diacetate group to yield DCFH which is oxidized by H2O2 to DCF and fluoresces green upon excitationOxidation by H2O2 yields rhodamine 123, which fluoresces blue upon excitationOxiVision™ probes are oxidized by intracellular H2O2. Generates fluorescence upon excitation.
Ex/Em (nm)504/525507/527405/450497/516
Filter SetFITCFITCDAPIFITC
Live CellsYes
ImagingYes
HCSYes
Flow CytometryYes
Unit Size25 mg10 mgKit (100 Tests)Kit (100 Tests)
Cat No.152041520611504 (Imaging)
11505 (Flow)
11503 (Imaging)
11506 (Flow)

 

Hydroxyl Radical


The detection of intracellular hydroxyl radical (•OH) is of central importance to the understanding of proper cellular redox regulation and the impact of its dysregulation on various pathologies. The hydroxyl radical is one of the reactive oxygen species (ROS) that are highly reactive with other molecules to achieve stability. In general, hydroxyl radicals are considered to be a harmful byproduct of oxidative metabolism, which can cause molecular damage in living systems. It shows an average lifetime of 10-9 nanoseconds and can react with nearly every biomolecule, such as nuclear DNA, mitochondrial DNA, proteins and membrane lipids.

Hydroxyl Radical Detection
 

Table 1. Intracellular ROS Selection Guide

ROS Species
ROS Brite™ 570
ROS Brite™ 670
ROS Brite™ 700
ROS Brite™ HDCF
Amplite® ROS Green
Amplite® ROS Red
Hydrogen Peroxide (H2O2)++++++++++++
Hydroxyl radical (•OH)+++++++++
Tert-butyl-hyrdoperoxide (TBHP)++++++
Hypochlorous acid (HOCl)-+++-+-
Superoxide anion (O2•-)+++++---
Nitric Oxide (NO)------
Peroxynitrite anion (ONOO-)------
Cat No.2290222903160041605322901

Table 2. ROS-Selective Probes and Assay Kits

ROS Species
OxiVision™ Green
OxiVision™ Blue
MitoROS™ 520
MitoROS™ 580
MitoROS™ OH580
Hydrogen Peroxide (H2O2)++++++---
Hydroxyl radical (•OH)----+++
Tert-butyl-hyrdoperoxide (TBHP)-----
Hypochlorous acid (HOCl)-----
Superoxide anion (O2•-)--++++++-
Nitric Oxide (NO)-----
Peroxynitrite anion (ONOO-)-----
Cat No.16060

 

Additional Resources


 

Table 3. ROS generation methods

Reactive Oxygen Specie (ROS)
ROS Generation Method
H2O2100 µM of H2O2
•O2-100 µM of KO2
1O2100 µM of 3-(1,4-dihydro-1,4-epidioxy-1-naphthyl)propionic acid
-OCL3 µM (final) of -OCl
•OH100 µM of ferrous perchlorate (II) and 1 mM of H2O2
ONOO-3 µM (final) of ONOO-
NO100 µM of 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-methyl-1-triazene
ROO•100 µM of 2,2'-azobis(2-amidinopropane), dihydrochloride (AAPH)
Auto-oxidation2.5 hours of exposure to a fluorescent light source

 

Product Ordering Information


 

Table 4. Ordering Information For Reactive Oxygen Species (ROS) Detecting Products

Cat No.
Product
Unit Size
15200Hydroethidine [Dihydroethidium] *CAS 104821-25-2*25 mg
15204DCFH-DA [2',7'-Dichlorodihydrofluorescein diacetate] *CAS 4091-99-0*25 mg
152201,2-Diaminoanthraquinone *CAS 1758-68-5*25 mg
152212,3-Diaminonaphthalene *CAS 771-97-1*100 mg
15250MCLA [2-Methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, hydrochloride] *CAS 128322-44-1*1 mg
15991Cell Meter™ Intracellular Colorimetric Lipid Peroxidation (MDA) Assay Kit200 Tests
16000ROS Brite™ 570 *Optimized for Detecting Reactive Oxygen Species (ROS)*1 mg
16002ROS Brite™ 670 *Optimized for Detecting Reactive Oxygen Species (ROS)*1 mg
16050ROS Brite™ APF *Optimized for Detecting Reactive Oxygen Species (ROS)*1 mg
16051ROS Brite™ HPF *Optimized for Detecting Reactive Oxygen Species (ROS)*1 mg
16052MitoROS™ 580 *Optimized for Detecting Reactive Oxygen Species (ROS) in Mitochondria*500 Tests
16290DAF-2 1 mg
16293DAF-2 DA 10x50 µg
16296DAF-FM 1 mg
16298DAF-FM diacetate 10x50 µg
16315Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit *Green Fluorescence*200 Tests
16317Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit *Optimized for Flow Cytometry*100 Tests
16350Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *Orange Fluorescence Optimized for Microplate Reader*200 Tests
16351Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *Orange Fluorescence Optimized for Flow Cytometry*100 Tests
16356Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *Red Fluorescence Optimized for Flow Cytometry*100 Tests
16359Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *NIR Fluorescence Optimized for Microplate Reader*200 Tests
16360Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *NIR Fluorescence Optimized for Flow Cytometry*100 Tests
21259Lucigenin [Bis-N-methylacridinium nitrate] 10 mg
21505OxiVision™ Green hydrogen peroxide sensor 1 mg
22906Cell Meter™ Fluorimetric Cellular Lipid Peroxidation Assay Kit200 Tests
23290Bodi Fluor™ 581/591 C11 *Lipid Peroxidation Probe*1 mg