Selecting the right ROS probe

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Latest News and Events: Update to Spectrum Viewer
Calbryte™ series now available
A practical guide for use of PE and APC in flow cytometry
Selecting the right ROS probe
A highly sensitive direct ELISA of cAMP without acetylation
Quantitative comparison of iFluor™ 488- and FITC- conjugated antibodies for use in cell labeling

AssayWise Letters 2017, Vol. 6(2)
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Reactive oxygen species (ROS) are chemically reactive, oxygen-containing chemical species that are generated as byproducts of cellular metabolism. In animal cells, generation of ROS typically occurs in the mitochondria. In plant cells, ROS can also be generated in chloroplasts. More recently, ROS has been shown to be generated in peroxisomes and from plasma membrane oxidases of various cell types.

Reduction of oxygen and its byproducts.

When generated in excess, ROS has long been thought to result in damage of cellular macromolecules such as DNA, lipids and proteins. Holistically, this is represented by a cell's oxidative stress state. Such states of stress have been linked to cellular processes such as apoptosis, and more macroscopically, play a role in the pathogenesis of many human diseases.

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.

While ROS has been extensively studied for their detrimental effect on cells, it is only more recently that studies have looked at the role of ROS in cell signaling. In controlled amounts, ROS has been shown to regulate gene activation. The specific mechanism by which this occurs is, however, still up for debate. It is possible that ROS binds to special receptors which initiate a signaling cascade leading up to gene regulation. It is also possible that ROS directly modifies the proteins in such a signaling cascade, perhaps by regulation of protein phosphorylation.

Due to its importance in biological systems, a plethora of tools have been developed to study ROS both in vitro and in vivo. The table below provides a summary of the most common tools as well as their targets.

Target	Application	Tools	Catalog Number	Principle
Hydrogen peroxide (H₂O₂) a product of many enzymatic ROS scavenging pathways. The most well studied is superoxide dismutase activity, which catalyzes the reduction of superoxide anion to hydrogen peroxide.	live cell	DCFH-DA	15204	Probe enters cell wherein esterases cleave off diacetate group. Then DCFH is oxidized by hydrogen peroxide to DCF and emits green fluorescence upon excitation.^[4]
		Dihydrorhodamine 123	15206, 15207	Probe passively permeates cell membrane. Oxidation by hydrogen peroxide yields rhodamine 123, which fluoresces blue upon excitation.
		OxiVision™ Blue	11504, 11505	Probe permeates cell and is oxidized by intracellular hydrogen peroxide. Generates fluorescence upon excitation.
		OxiVision™ Green	11503, 11506
	cell extract; solutions	Amplite^® Fluorimetric Hydrogen Peroxide Assay Kit (Red)	11501	Hydrogen peroxide dependent oxidation of ADHP (synonyms: 10-acetyl-3,7-dihydroxyphenoxazine, Amplex^® Red) by horseradish peroxidase (HRP) converts ADHP to resorufin. Resorufin can be detected using colorimetric or fluorimetric methods.
		Amplite^® Colorimetric Hydrogen Peroxide Assay Kit	11500
		Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit	Not available
		Amplite^® Fluorimetric Hydrogen Peroxide Assay Kit (Infrared)	11502	Hydrogen peroxide dependent oxidation of Amplite IR by horseradish peroxidase (HRP) generates activated probe. Excitation emits a near-infrared fluorescent signal
Superoxide anion (O₂^-) a by-product of aerobic metabolism, such as mitochondrial respiration (particularly Complex I and Complex III).	live cell; cell extract; solutions	Lucigenin	21259	Lucigenin is activated by conversion to lucigenin cation radical. Lucigenin cation radical reacts with superoxide anion to produce dioxetane intermediate, which decomposes to N-methylacridone. High energy electrons in N-methylacridone fall to lower energy state, resulting in luminescence.^[5]
		Coelenterazine	21150	Oxidation by superoxide anion results in an excited electron state. Upon decay to ground state electron configuration, photons are released as luminescence.^[8]
		Luminol	11050	Superoxide-dependent enzyme catalyzed oxidation of luminol results in luminescence.^[6]
		Hydroethidine	15200	Probe passively permeates intact cells and localizes in the mitochondria. Probe is activated through oxidation by superoxide. The activated probe intercalates with DNA and, upon excitation, fluoresces. DNA binding is necessary for strong fluorescence signal. For hydroethidine, activated probe is ethidium (ie. same active species as DNA stain ethidium bromide).^[3,7,12]
		MitoSox™ Red	Not available
		MitoROS™ 580	16052, 22970, 22971
		MitoROS™ 520	16060	Probe readily passes through intact cell membranes whereupon it localizes in mitochondria. It is then oxidized by superoxide. Upon excitation, it releases a green fluorescence.
Hydroxyl radical (•OH) can be generated when superoxide anions react with transition metals. Extremely reactive. Can react with hydrogen on DNA backbone, resulting in strand breakage.	live cell; cell extract; solutions	MitoROS™ OH580	16055	Probe is able to freely enter live cells wherein it becomes oxidized specifically by free hydroxyl radicals. Upon excitation, oxidized probe fluoresces red.
Total ROS includes hydrogen peroxide, superoxide anion, hydroxyl radical, singlet oxygen, nitric oxide, butyl peroxide and hypochlorous acid	live cell; cell extract; solutions	ROS™ Brite 570	16000, 22902	Probe passively permeates intact cell membranes. Once inside the cell, probe is oxidized by intracellular ROS. Probe can also be oxidized by ROS in solution for cell extract assays. Upon excitation, probe emits a fluorescent signal.
		ROS™ Brite 670	16002, 22901
		ROS™ Brite 700	16004, 22903
		Amplite^® ROS Green	22900

Target	Tool	Catalog Number	Principle
SOD dismutase (SOD) catalyzes conversion (ie. dismutation) of superoxide into oxygen and hydrogen peroxide	Amplite^® Colorimetric Superoxide Dismutase (SOD) Assay Kit	11305	First uses xanthine oxidase (XO) to convert xanthine into hydrogen peroxide and uric acid while simultaneously catalyzing the reduction of molecular oxygen (O₂) into superoxide anion (O₂^-). Then uses competitive inhibition of superoxide dismutation by ReadiView™ SOD560 to quantify superoxide dismutase activity. Decrease in absorbance of ReadiView™ SOD560 is directly proportional to SOD activity.
Catalase nearly ubiquitous antioxidant enzyme that converts hydrogen peroxide to water and molecular oxygen	Amplite^® Fluorimetric Catalase Assay Kit (Red)	11306	Competitive inhibition assay. Amplite^® Red probe is activated through oxidation by hydrogen peroxide. Thus, probe competes with catalase for hydrogen peroxidase substrate. Amplite Red absorbance is inversely proportional to catalase activity.
Glutathione (GSH) very important redox compound. It is a major component of the glutathione-ascorbate cycle which converts hydrogen peroxide into water	Thiolite™ Green	10055, 22810	Probe becomes activated after reaction with glutathione. Upon excitation, probe releases a green fluorescence.
	Amplite^® Fluorimetric Glutathione GSH/GSSG Ratio Assay Kit	10056, 10060	Uses Thiolite™ Green probe, which becomes activated after reaction with glutathione (GSH), to quantify glutathione. Oxidized glutathione (GSSG) is determined by measuring GSH concentration before and after enzyme-catalyzed reduction of GSSG to GSH. GSSG concentration is calculated by subtracting initial GSH (before enzyme reaction) from total GSH (after enzyme reaction).
Glutathione peroxidase (GPx) catalyzes the oxidation of GSH to GSSG and the conversion of hydrogen peroxide to water	Amplite^® Fluorimetric Glutathione Peroxidase Assay Kit (Blue)	11560	Enzymatic cycling assay. Glutathione peroxidase (GPx) catalyzes the oxidation of glutathione from GSH to GSSG. Glutathione reductase (GR) then catalyzes the reduction of GSSG back into GSH with the coenzyme NADPH, which is oxidized to NADP+. The Quest Fluor™ NADP Probe then quantifies the level of NADP+ which is directly proportional to the original GPx activity.
Ascorbate also called ascorbic acid or vitamin C, it is a major component of the glutathione-ascorbate cycle which converts hydrogen peroxide to water	Amplite^® Fluorimetric Ascorbic Acid Assay Kit (Blue)	13835	Relies on the dehydrogenation of ascorbic acid to dehydroascorbic acid (DHA) by an enzyme-catalyzed reaction. Resulting DHA is quantified by Ascorbrite Blue probe.
NAD+/NADH major component in ATP synthesis that occurs in mitochondria. This coenzyme is thought to be involved in superoxide production, the rate of which is dependent on the NAD+/NADH ratio.	Amplite^® NAD/NADH Kits (assorted)	15273, 15258, 15275, 15280, 15263, 15257, 15261, 15262, 15291, 15290, 15259, 15271	Typically, uses a NAD+ specific probe to quantify NAD+ concentration. NAD+/NADH ratio is determined by enzymatic cycling assay.
NADPH acts as an electron donor in the glutathione-ascorbate cycle that converts hydrogen peroxide to water	Amplite^® NADPH Kits (assorted)	15274, 15272, 15260, 15276, 15264, 15262, 15259, 15291, 15290	Typically, uses a NADPH specific probe to quantify NADPH concentration. NADP/NADPH ratio is determined by enzymatic cycling assay.

References

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Bindokas, Vytautas P., et al. "Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine."Journal of Neuroscience4 (1996): 1324-1336.
Kalyanaraman, Balaraman, et al. "Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations."Free Radical Biology and Medicine1 (2012): 1-6.
LeBel, Carl P., Harry Ischiropoulos, and Stephen C. Bondy. "Evaluation of the probe 2', 7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress."Chemical research in toxicology2 (1992): 227-231.
Li, Yunbo, et al. "Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems."Journal of Biological Chemistry4 (1998): 2015-2023.
Misra, Hara P., and Pamela M. Squatrito. "The role of superoxide anion in peroxidase-catalyzed chemiluminescence of luminol."Archives of biochemistry and biophysics1 (1982): 59-65.
Mukhopadhyay, Partha, et al. "Simple quantitative detection of mitochondrial superoxide production in live cells."Biochemical and biophysical research communications1 (2007): 203-208.
Münzel, Thomas, et al. "Detection of superoxide in vascular tissue."Arteriosclerosis, thrombosis, and vascular biology11 (2002): 1761-1768.
Murphy, Michael P. "How mitochondria produce reactive oxygen species."Biochemical Journal1 (2009): 1-13.
Thannickal, Victor J., and Barry L. Fanburg. "Reactive oxygen species in cell signaling."American Journal of Physiology-Lung Cellular and Molecular Physiology6 (2000): L1005-L1028.
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Zielonka, Jacek, and B. Kalyanaraman. "Hydroethidine-and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth."Free Radical Biology and Medicine48.8 (2010): 983-1001.

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