Generating and Detecting Reactive Oxygen Species—Section 18.2

We offer an assortment of Molecular Probes products for the generation of reactive oxygen species (ROS), including singlet oxygen (¹O₂), superoxide (·O₂^–), hydroxyl radical (HO·) and various peroxides (ROOR') and hydroperoxides (ROOH) (Reactive oxygen species—Table 18.1), as well as for their fluorometric detection in solution. Although there are no equilibrium sensors that continuously monitor the level of reactive oxygen species, this section discusses a number of probes that trap or otherwise react with singlet oxygen, hydroxyl radicals or superoxide. The optical or electron spin properties of the resulting products can be used as a measure of the presence or quantity of the reactive oxygen species and, in certain cases, can report the kinetics and location of their formation.

Singlet oxygen is responsible for much of the physiological damage caused by reactive oxygen species, including nucleic acid modification through selective reaction with deoxyguanosine. The lifetime of singlet oxygen is sufficiently long (4.4 microseconds in water ) to permit significant diffusion in cells and tissues. In the laboratory, singlet oxygen is usually generated in one of three ways: photochemically from dioxygen (³O₂) using a photosensitizing dye; chemically by thermal decomposition of a peroxide or dioxetane; or by microwave discharge through an oxygen stream. The generation of singlet oxygen can also be targeted to the mitochondria by several cationic dyes. Singlet oxygen can be detected by its characteristic weak chemiluminescence at 1268 nm or at 634 and 703 nm.

Hypericin

Among the most efficient reagents for generating singlet oxygen is the photosensitizer hypericin (H7476, ), a natural pigment isolated from plants of the genus Hypericum. This heat-stable dye exhibits a quantum yield for singlet oxygen generation in excess of 0.7, as well as high photostability, making it an important agent for both anticancer and antiviral research. Hypericin is also an effective inhibitor of both protein kinase C and tyrosine protein kinase (Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3).

Because its chemical reactivities are well characterized, hypericin is amenable to conjugation to a variety of primary and secondary detection reagents. Not only does this photosensitizing dye efficiently oxidize diaminobenzidine (DAB) to form an insoluble, electron-dense DAB oxidation product (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2), but it exhibits modest fluorescence quantum yields and broad UV and visible spectra. Thus, hypericin-labeled detection reagents are compatible with fluorescence, light or electron microscopy applications.

Rose Bengal Diacetate

Rose bengal diacetate (R14000) is an efficient, cell-permeant generator of singlet oxygen. It is an iodinated xanthene derivative that has been chemically modified by the introduction of acetate groups (). These modifications inactivate both its fluorescence and photosensitization properties, while increasing its ability to cross cell membranes. Once inside a live cell, esterases remove the acetate groups, restoring rose bengal to its native structure. Its intracellular localization allows rose bengal diacetate to be a very effective photosensitizer.

Merocyanine 540

Photolysis of merocyanine 540 (M24571, ) produces both singlet oxygen and other reactive oxygen species, including oxygen radicals. Merocyanine 540 is commonly used as a photosensitizer in photodynamic therapy.

Halogenated Fluoresceins

Halogenated derivatives of fluorescein dyes are known to be effective photosensitizers and singlet oxygen generators. This property of eosin and other halogenated fluoresceins and their conjugates has been exploited to improve ultrastructural resolution (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2). Bioconjugates of eosin can be prepared using the reactive derivatives described in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1 and Thiol-Reactive Probes—Chapter 2.

Singlet Oxygen Sensor Green Reagent

Unlike other fluorescent and chemiluminescent singlet oxygen detection reagents, the Singlet Oxygen Sensor Green reagent (S36002) is highly selective for singlet oxygen (¹O₂); it shows no appreciable response to other ROS, including hydroxyl radical (HO·), superoxide (·O₂^–) and nitric oxide (NO) (Figure 18.2). Before reaction with singlet oxygen, this probe initially exhibits weak blue fluorescence with excitation peaks at 372 and 393 nm and emission peaks at 395 and 416 nm. In the presence of singlet oxygen, however, it emits a green fluorescence similar to that of fluorescein (excitation/emission maxima ~504/525 nm).

We have observed that the fluorescent product of Singlet Oxygen Sensor Green reagent can degrade with time in some solutions and that Singlet Oxygen Sensor Green reagent can become fluorescent at alkaline pH in the absence of singlet oxygen. Nevertheless, with the proper controls the intensity of the green-fluorescent signal can be correlated with singlet oxygen concentration, without significant interference from other ROS. The Singlet Oxygen Sensor Green reagent is available as a cell-impermeant derivative (S36002) for detecting singlet oxygen in solution. It can also potentially serve to assess the efficacy of free radical scavengers, which are frequently used to improve the flavor and nutritional quality in foods.

trans-1-(2'-Methoxyvinyl)pyrene

trans-1-(2'-Methoxyvinyl)pyrene (M7913) can be used to detect picomole quantities of singlet oxygen in chemical and biological systems (Figure 18.3), making this compound one of the most sensitive singlet oxygen probe currently available. Furthermore, this highly selective chemiluminescent probe does not react with other activated oxygen species such as hydroxyl radical, superoxide or hydrogen peroxide.

Hydroxyl and superoxide radicals have been implicated in a number of pathological conditions, including ischemia, reperfusion and aging. The superoxide anion (Reactive oxygen species—Table 18.1) may also play a role in regulating normal vascular function. The hydroxyl radical is a very reactive oxygen species that has a lifetime of about 2 nanoseconds in aqueous solution and a radius of diffusion of about 20 Å. Thus, it induces peroxidation only when it is generated in close proximity to its target. The hydroxyl radical can be derived from superoxide in a reaction catalyzed by Fe²⁺ or other transition metals, as well as by the effect of ionizing radiation on dioxygen. Superoxide is most effectively generated from a hypoxanthine–xanthine oxidase generating system. In one cell-based assay for hydroxyl radical formation, D-phenylalanine is specifically converted to D-tyrosine. In phagocytic cells, H₂O₂ also produces O,O'-dityrosine, an oxidative crosslink product of appropriately situated tyrosine residues, which is formed through the intermediacy of phenoxyl radicals. O,O'-Dityrosine is intrinsically fluorescent at a relatively high pH or can be detected using specific antibodies.

Malachite Green

Malachite green is a nonfluorescent photosensitizer that absorbs at long wavelengths (~630 nm). Its photosensitizing action can be targeted to particular cellular sites by conjugating malachite green isothiocyanate (M689, ) to specific antibodies. Enzymes and other proteins within ~10 Å of the binding site of the malachite green–labeled antibody can then be selectively destroyed upon irradiation with long-wavelength light. Studies by Jay and colleagues have demonstrated that this photoinduced destruction of enzymes in the immediate vicinity of the chromophore is apparently the result of localized production of hydroxyl radicals, which have short lifetimes that limit their diffusion from the site of their generation.

1,10-Phenanthroline Iodoacetamide

Conjugation of the iodoacetamide of 1,10-phenanthroline (P6879, ) to thiol-containing ligands confers the metal-binding properties of this important complexing agent on the ligand. For example, the covalent copper–phenanthroline complex of oligonucleotides or nucleic acid–binding molecules in combination with hydrogen peroxide acts as a chemical nuclease to selectively cleave DNA or RNA. Hydroxyl radicals or other reactive oxygen species appear to be involved in this cleavage.

MitoSOX Red Mitochondrial Superoxide Indicator

Mitochondrial superoxide is generated as a by-product of oxidative phosphorylation. In an otherwise tightly coupled electron transport chain, approximately 1–3% of mitochondrial oxygen consumed is incompletely reduced; these "leaky" electrons can quickly interact with molecular oxygen to form superoxide anion, the predominant ROS in mitochondria. Increases in cellular superoxide production have been implicated in cardiovascular diseases, including hypertension, atherosclerosis and diabetes-associated vascular injuries, as well as in neurodegenerative diseases such as Parkinson disease, Alzheimer disease and amyotrophic lateral sclerosis (ALS).

The assumption that mitochondria serve as the major intracellular source of ROS has been based largely on experiments with isolated mitochondria rather than direct measurements in live cells. MitoSOX Red mitochondrial superoxide indicator (M36008) is a novel fluorogenic dye for highly selective detection of superoxide in the mitochondria of live cells (). MitoSOX Red reagent is live-cell permeant and is rapidly and selectively targeted to the mitochondria. Once in the mitochondria, MitoSOX Red reagent is oxidized by superoxide and exhibits bright red fluorescence upon binding to nucleic acids (excitation/emission maxima = 510/580 nm). MitoSOX Red reagent is readily oxidized by superoxide but not by other ROS- or reactive nitrogen species (RNS)–generating systems, and oxidation of the probe is prevented by superoxide dismutase (Figure 12.20). This reagent may enable researchers to distinguish artifacts of isolated mitochondrial preparations from direct measurements of superoxide generated in the mitochondria of live cells. MitoSOX Red mitochondrial superoxide indicator can also be used as a tool in the discovery of agents that modulate oxidative stress in various pathologies.

Fluorogenic Spin Traps

Hydroxyl radicals have usually been detected after reaction with spin traps. We offer TEMPO-9-AC (A7923, ) and proxyl fluorescamine (C7924, ), two fluorogenic probes for detecting hydroxyl radicals and superoxide. Each of these molecules contains a nitroxide moiety that effectively quenches its fluorescence. However, once TEMPO-9-AC or proxyl fluorescamine traps a hydroxyl radical or superoxide, its fluorescence is restored and the radical's electron spin resonance signal is destroyed, making these probes useful for detecting radicals either by fluorescence or by electron spin resonance spectroscopy. TEMPO-9-AC has been reported to detect glutathionyl radicals but not phenoxyl radicals. Proxyl fluorescamine can be used to detect the methyl radicals that are formed by reacting hydroxyl radicals with DMSO. Radical-specific scavengers—such as the superoxide-specific p-benzoquinone and superoxide dismutase or the hydroxyl radical–specific mannitol and dimethylsulfoxide (DMSO) —can be used to identify the detected species.

Chemiluminescent and Chromogenic Reagents for Detecting Superoxide

In the absence of apoaequorin, the luminophore coelenterazine (C2944) produces chemiluminescence in response to superoxide generation in phorbol ester– or chemotactic peptide–stimulated neutrophils. Unlike luminol, coelenterazine exhibits luminescence that does not depend on the activity of cell-derived myeloperoxidase and is not inhibited by azide.

In addition to coelenterazine, we offer MCLA (M23800, ) for detecting superoxide. MCLA and coelenterazine are superior alternatives to lucigenin (L6868) for this application because lucigenin can reportedly sensitize superoxide production, leading to false-positive results. An additional advantage of MCLA is that its pH optimum for luminescence generation is closer to the physiological near-neutral range than are the pH optima of luminol and lucigenin.

Lucigenin (L6868) exhibits chemiluminescence that is reported to be sensitive to the superoxide anion. Lucigenin has been employed to investigate superoxide generation in spermatozoa, L929 cells, chondrocytes contained within the matrix of live cartilage tissue, and mitochondria of alveolar macrophages. It has also been used to examine the role of the superoxide anion in reoxygenation injury in isolated rat hepatocytes. We have purified our lucigenin to remove a blue-fluorescent impurity that is found in some commercial samples.

Nitro blue tetrazolium salt (NBT, N6495; Tetrazolium salts for detecting redox potential in living cells and tissues—Table 18.2) and other tetrazolium salts are chromogenic probes useful for superoxide determination. These probes are also widely used for detecting redox potential of cells for viability, proliferation and cytotoxicity assays (see below).

In peroxisomes, H₂O₂ is produced by several enzymes that use molecular oxygen to oxidize organic compounds. This H₂O₂ is then used by catalase to oxidize other substrates, including phenols, formic acid, formaldehyde and alcohol. In liver and kidney cells, these oxidation reactions are important for detoxifying a variety of compounds in the bloodstream. However, H₂O₂ also plays a role in neurodegenerative and other disorders through induction of apoptosis and DNA strand breaks, modification of intracellular Ca²⁺ levels and mitochondrial potential, and oxidation of glutathione. In addition, H₂O₂ is released from cells during hypoxia.

Peroxidation of unsaturated lipids affects cell membrane properties, signal transduction pathways, apoptosis and the deterioration of foods and other biological compounds. Lipid hydroperoxides have been reported to accumulate in oxidatively stressed individuals, including HIV-infected patients. Lipid peroxidation may also be responsible for aging, as well as for pathological processes such as drug-induced phototoxicity and atherosclerosis, and is often the cause of free radical–mediated damage in cells. To directly assess the extent of lipid peroxidation, researchers either measure the amount of lipid hydroperoxides directly or detect the presence of secondary reaction products (e.g., 4-hydroxy-2-nonenal or malonaldehyde; see below). A problem with investigating the link between lipid peroxidation and diseases such as atherosclerosis, diabetes and Parkinson disease has been the lack of suitable methods to detect the relationship between lipid peroxidation and the onset of such diseases.

Peroxyl radicals are formed by the decomposition of various peroxides and hydroperoxides, including lipid hydroperoxides. The hydroperoxyl radical is also the protonated form of superoxide, and approximately 0.3% of the superoxide in the cytosol is present as this protonated radical. Experimentally, peroxyl radicals, including alkylperoxyl (ROO·) and hydroperoxyl (HOO·) radicals, are generated from compounds such as 2,2'-azobis(2-amidinopropane) and from hydroperoxides such as cumene hydroperoxide.

cis-Parinaric Acid

Fluorescence quenching of the fatty acid analog cis-parinaric acid (P36005) has been used in several lipid peroxidation assays, including quantitative determinations in live cells. In a study investigating the membrane antioxidant properties of the bcl-2 proto-oncogene product, researchers used cis-parinaric acid to detect lipid hydroperoxides together with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C2938) to detect cytosolic reactive oxygen molecules. Parinaric acid's extensive unsaturation () makes it quite susceptible to oxidation if not rigorously protected from air. Consequently, we offer cis-parinaric acid in a 10 mL unit size of a 3 mM solution in deoxygenated ethanol (P36005); if stored protected from light under an inert argon atmosphere at -20°C, this stock solution should be stable for at least six months. During experiments, we advise handling parinaric acid samples under inert gas and preparing solutions using degassed buffers and solvents. Parinaric acid is also somewhat photolabile and undergoes photodimerization when exposed to intense illumination, resulting in loss of fluorescence.

Diphenyl-1-Pyrenylphosphine

Hydroperoxides in lipids, serum, tissues and foodstuffs can be directly detected using the fluorogenic reagent diphenyl-1-pyrenylphosphine (DPPP, D7894). DPPP is essentially nonfluorescent until oxidized to a phosphine oxide by peroxides; in vitro, DPPP remains nonfluorescent in the presence of hydroxyl radicals generated by the Cu²⁺-ascorbated method. DPPP has previously been used to detect picomole levels of hydroperoxides by HPLC. Its solubility in lipids makes DPPP quite useful for detecting hydroperoxides in the membranes of live cells and in low-density lipoprotein particles, as well as for studying superoxide dismutase (SOD), which catalyzes the conversion of superoxide to hydrogen peroxide. In U937 cells, exposure to DPPP did not appreciably affect cell viability, proliferation or morphology for three days, and the fluorescent phosphine oxide was stable in the cell membranes for at least two days.

BODIPY Dyes: Peroxyl Radical Scavengers

The BODIPY 581/591 fatty acid (D3861) is a sensitive fluorescent reporter for lipid peroxidation, shifting from red to green fluorescence in the presence of both reactive oxygen and reactive nitrogen species. The oxidation and nitroxidation products of this BODIPY fatty acid have been characterized by mass spectrometry. Also using mass spectrometry analysis of oxidation products, MacDonald and co-workers report that BODIPY 581/591 fatty acid is more sensitive to oxidation than endogenous lipids, and therefore tends to overestimate oxidative damage and underestimate antioxidant protection effects. They suggest that while BODIPY 581/591 fatty acid should not be used to quantitate lipid oxidation, it remains a sensitive indicator of free radical processes in lipid membranes.

A unique assay for peroxyl radicals uses BODIPY dyes, including the BODIPY 581/591 fatty acid and the BODIPY 665/676 dye (B3932), to measure antioxidant activity in an organic lipid environment or in a liposomal medium. This assay is based on the loss or shift of the dye's fluorescence as the result of its interaction with peroxyl radicals, and on the retention of the fluorescent signal in the presence of antioxidants that intercept these free radicals. It has been proposed that this assay is suitable for examining the effect of lipid peroxidation on a cell-by-cell basis using a fluorescence microplate reader or flow cytometer.

The oxidation sensitivity of the conjugated double bonds of the BODIPY 581/591 fatty acid () has also been exploited in a ratiometric assay of lipid oxidation in live cells that is essentially independent of uneven dye loading, cell thickness and compartmentalization. In this assay, the cell membranes of rat-1 fibroblasts and myocardial cells were labeled with the BODIPY 581/591 fatty acid, and then cumene hydroperoxide was added to induce oxidation. Upon oxidation, the BODIPY 581/591 fluorophore exhibited a shift in its fluorescence from red to green, which was observed using confocal laser-scanning microscopy. As compared with arachidonic acid, the fluorescence of this BODIPY probe was reportedly twice as sensitive to oxidation. In a similar application, the BODIPY 581/591 fatty acid was used to detect lipid peroxidation in equine spermatozoa after exposure to ferrous sulfate, sodium ascorbate, cumene hydroperoxide and cooled storage, and ratiometric measurements were made with either a fluorescence microplate reader or a flow cytometer.

Peroxyl radicals have also been detected in erythrocyte and red blood cell membranes using BODIPY FL EDA (D2390, Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4), a water-soluble BODIPY dye, or BODIPY FL hexadecanoic acid (D3821, Fatty Acid Analogs and Phospholipids—Section 13.2). BODIPY FL hexadecanoic acid exhibits the red shift common to the fluorescence of lipophilic BODIPY dyes when they are concentrated, permitting ratiometric measurements of hydroxyl radical production and allowing the onset of lipid peroxidation in live cells to be monitored.

Other Scavengers for Peroxyl Radicals

The fluorescence of several other probes is lost following interaction with peroxyl radicals. Lipophilic fluorescein dyes such as hexadecanoylaminofluorescein (H110, Other Nonpolar and Amphiphilic Probes—Section 13.5) and fluorescein-labeled phosphatidylethanolamine (F362, Fatty Acid Analogs and Phospholipids—Section 13.2) have been useful for detecting peroxyl radical formation in membranes and in solution. Phycobiliproteins such as B-phycoerythrin, R-phycoerythrin and allophycocyanin (P800, P801, A803, A819; Phycobiliproteins—Section 6.4) may be similarly useful. R-phycoerythrin has been used to detect and measure total plasma antioxidant capacity, including peroxyl radicals.

Luminol

Although luminol (L8455) is not useful for detecting superoxide in live cells, it is commonly employed to detect peroxidase- or metal ion–mediated oxidative events. Used alone, luminol can detect oxidative events in cells rich in peroxidases, including granulocytes and spermatozoa. This probe has also been used in conjunction with horseradish peroxidase (HRP) to investigate reoxygenation injury in rat hepatocytes. In these experiments, it is thought that the primary species being detected is hydrogen peroxide. In addition, luminol has been employed to detect peroxynitrite, a molecule thought to be generated in a variety of pathological conditions. Phospholipid hydroperoxides have been determined directly by chemiluminescence-detected HPLC with luminol or a combination of luminol and cytochrome c. This chemiluminescent probe has also been employed as a chemical sensor for dioxygen and nitrogen dioxide. It has been reported that luminol's chemiluminescence response to oxidative species may be competitively inhibited by biomolecules containing sulfhydryl and thioether groups.

Detecting 4-Hydroxy-2-Nonenal

Formation of 4-hydroxy-2-nonenal from linoleic acid is a major cause of lipid peroxidation–induced liver toxicity. Several reagents for the direct fluorometric detection of aldehydes are described in Reagents for Modifying Aldehydes and Ketones—Section 3.3. The modification of 4-hydroxy-2-nonenal or malonaldehyde with a fluorescent or chromophoric hydrazine or hydroxylamine reagent coupled with the separation and detection of the reaction product by a chromatography-based technique has rarely been reported but appears to be one of the most promising approaches for detecting these lipid peroxidation products.

Amplex Red Reagent: Stable Substrate for Peroxidase Detection

In the presence of horseradish peroxidase (HRP), Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine, A12222, A22177; ) reacts with H₂O₂ in a 1:1 stoichiometry to produce highly fluorescent resorufin (R363, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1, Figure 10.59). Amplex Red reagent has greater stability, yields less background and produces a red-fluorescent product that is more readily detected than the similar reduced methylene blue derivatives commonly used for colorimetric determination of lipid peroxides in plasma, sera, cell extracts and a variety of membrane systems. Using Amplex Red reagent in conjunction with HRP, we have found that release of H₂O₂ to the medium by as few as 2000 phorbol ester–stimulated neutrophils can be detected in a fluorescence microplate reader.

Amplex Red reagent has been used to detect the release of H₂O₂ from activated human leukocytes, to measure the activity of monoamine oxidase in cow brain tissue, to demonstrate the extracellular production of H₂O₂ produced by UV light stimulation of human keratinocytes and to measure L-glutamate in food samples. Using Amplex Red reagent, researchers have discovered that antibodies can convert molecular oxygen to H₂O₂, which may be important in understanding a new chemical arm of the immune system, as well as the evolution of antibodies and the role they may play in human diseases. Amplex Red reagent is available in a single 5 mg vial (A12222) or packaged as a set of 10 vials, each containing 10 mg of the substrate, for high-throughput screening applications (A22177).

Amplex UltraRed Reagent: Brighter and More Sensitive than the Amplex Red Reagent

Amplex UltraRed reagent (A36006) improves upon the performance of Amplex Red reagent, offering brighter fluorescence and enhanced sensitivity on a per-mole basis in horseradish peroxidase or horseradish peroxidase-coupled enzyme assays (Figure 10.62). Fluorescence of oxidized Amplex UltraRed reagent is also less sensitive to pH (Figure 10.63), and the substrate and its oxidation product exhibit greater stability than Amplex Red reagent in the presence of H₂O₂ or thiols such as dithiothreitol (DTT). Like Amplex Red reagent, nonfluorescent Amplex UltraRed reagent reacts with H₂O₂ in a 1:1 stoichiometric ratio to produce a brightly fluorescent and strongly absorbing reaction product (excitation/emission maxima ~568/581 nm) (). Because the reaction product has long-wavelength spectra, there is little interference from the blue or green autofluorescence found in most biological samples.

Amplex UltraRed reagent can provide increased sensitivity in peroxidase-based enzyme-linked immunosorbent assays (ELISAs). With a high extinction coefficient and good quantum efficiency, the fluorescence-based Amplex UltraRed reagent is more sensitive than standard colorimetric reagents and provides a broader measurement range for ELISAs. In contrast to commonly used ELISA reagents such as ABTS and TMB, Amplex UltraRed reagent is exceptionally resistant to autooxidation, making it a superior alternative for peroxidase detection (Advantages of the Amplex UltraRed reagent over chromogenic reagents—Table 10.5). Like Amplex Red reagent, the versatile Amplex UltraRed reagent can be detected using either fluorescence- or absorption-based instrumentation. Amplex UltraRed reagent, which should be suitable for any of the applications described for Amplex Red reagent (Substrates for Oxidases, Including Amplex Red Kits—Section 10.5), is available as a set of five vials, each containing 1 mg of the substrate (A36006).

Figure 10.62 Detection of H2O2 using Amplex UltraRed reagent (red square) or Amplex Red reagent (blue triangle). Reactions containing 50 µM Amplex UltraRed or Amplex Red reagent, 1 U/mL HRP and the indicated amount of H2O2 in 50 mM sodium phosphate buffer, pH 7.4, were incubated for 30 minutes at room temperature. The inset shows the sensitivity and linearity of the Amplex UltraRed assay at low levels of H2O2.

Figure 10.63 Comparison of pH-dependent fluorescence of Amplex UltraRed reagent (solid blue circles) and Amplex Red reagent (open blue squares). Fluorescence intensities were measured using excitation/emission of ~570/585 nm.

Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit

The Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188) provides a simple, sensitive, one-step assay for detecting H₂O₂ or the activity of horseradish peroxidase either by measuring fluorescence with a fluorescence-based microplate reader or a fluorometer (Figure 10.66) or by measuring absorption with an absorption-based microplate reader or a spectrophotometer. The Amplex Red peroxidase substrate can detect the presence of active peroxidases and the release of H₂O₂ from biological samples, including cells and cell extracts and is also useful for detecting H₂O₂ that is produced as a product of enzyme-coupled reactions. The Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxide (HRP)
• H₂O₂ for use as a positive control
• Concentrated reaction buffer
• Detailed protocols (Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit)

Each kit provides sufficient reagents for approximately 500 assays using a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay. Several additional kits that utilize the Amplex Red peroxidase substrate to detect H₂O₂ in coupled enzymatic reactions are described in Substrates for Oxidases, Including Amplex Red Kits—Section 10.5.

Figure 10.66 Detection of HRP using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188). Reactions containing 50 µM Amplex Red reagent, 1 mM H2O2 and the indicated amount of HRP in 50 mM sodium phosphate buffer, pH 7.4, were incubated for 30 minutes at room temperature. Fluorescence was measured with a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. Background fluorescence (3 units), determined for a no-HRP control reaction, was subtracted from each value. The inset shows the sensitivity of the assay at very low levels of HRP.

Amplex Red Xanthine/Xanthine Oxidase Assay Kit

Xanthine oxidase (E.C. 1.2.3.2) plays a key role in the production of free radicals, including superoxide, in the body. The Amplex Red Xanthine/Xanthine Oxidase Assay Kit (A22182) provides an ultrasensitive method for detecting xanthine or hypoxanthine or for monitoring xanthine oxidase activity. In the assay, xanthine oxidase catalyzes the oxidation of purine nucleotides, hypoxanthine or xanthine, to uric acid and superoxide. In the reaction mixture, the superoxide spontaneously degrades to H₂O₂, which in the presence of HRP reacts stoichiometrically with Amplex Red reagent to generate the red-fluorescent oxidation product, resorufin. Resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm (), respectively, and because the extinction coefficient is high (54,000 cm^-1M^-1), the assay can be performed either fluorometrically or spectrophotometrically.

The Amplex Red Xanthine/Xanthine Oxidase Assay Kit (A22182) contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• H₂O₂
• Concentrated reaction buffer
• Xanthine oxidase from buttermilk
• Hypoxanthine
• Xanthine
• Detailed protocols (Amplex Red Xanthine/Xanthine Oxidase Assay Kit)

Each kit provides sufficient reagents for approximately 400 assays using either a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay.

In healthy individuals, xanthine oxidase is present in appreciable amounts only in the liver and jejunum. In various liver disorders, however, the enzyme is released into circulation. Therefore, determination of serum xanthine oxidase levels serves as a sensitive indicator of acute liver damage such as jaundice. The Amplex Red xanthine/xanthine oxidase assay has been used as a marker of recovery from exercise stress. Previously, researchers have utilized chemiluminescence or absorbance to monitor xanthine oxidase activity. The Amplex Red Xanthine/Xanthine Oxidase Assay Kit permits the detection of xanthine oxidase in a purified system at levels as low as 0.1 mU/mL by fluorescence (Figure 18.4). This kit can also be used to detect as little as 200 nM hypoxanthine or xanthine (Figure 18.5), and, when coupled to the purine nucleotide phosphorylase enzyme, to detect inorganic phosphate.

Figure 18.4 Detection of xanthine oxidase using the Amplex Red Xanthine/Xanthine Oxidase Assay Kit (A22182). Each reaction contained 50 µM Amplex Red reagent, 0.2 U/mL horseradish peroxidase, 0.1 mM hypoxanthine and the indicated amount of xanthine oxidase in 1X reaction buffer. After 30 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and detection at 590 ± 17.5 nm. A background of 65 fluorescence units was subtracted from each data point. The inset shows the assay’s sensitivity and linearity at low hypoxanthine concentrations.

Figure 18.5 Detection of hypoxanthine using the Amplex Red Xanthine/Xanthine Oxidase Assay Kit (A22182). Each reaction contained 50 µM Amplex Red reagent, 0.2 U/mL horseradish peroxidase, 20 mU/mL xanthine oxidase and the indicated amount of hypoxanthine in 1X reaction buffer. Reactions were incubated at 37°C. After 30 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and detection at 590 ± 17.5 nm. A background of 54 fluorescence units was subtracted from each data point. The inset shows the assay’s sensitivity and linearity at low enzyme concentrations.

EnzChek Myeloperoxidase (MPO) Activity Assay Kit

Myeloperoxidase (MPO, EC 1.11.1.7) is a lysosomal hemeoprotein located in the azurophilic granules of polymorphonuclear (PMN) leukocytes and monocytes. It is a dimeric protein composed of two 59 kD and two 13.5 kD subunits. MPO is a unique peroxidase that catalyzes the conversion of hydrogen peroxide (H₂O₂) and chloride to hypochlorous acid, the major strong oxidant with powerful antimicrobial activity and broad-spectrum reactivity with biomolecules. MPO is considered an important marker for inflammatory diseases, autoimmune diseases and cancer. MPO is also experimentally and clinically important for distinguishing myeloid from lymphoid leukemia and, due to its role in the pathology of atherogenesis, has been advocated as a prognostic marker of cardiovascular disease.

The ferric, or native, MPO reacts with hydrogen peroxide (H₂O₂) to form the active redox and enzyme intermediate compound MPO-I, which oxidizes chloride (Cl^–) to HOCl; these reactions make up the chlorination cycle. MPO also oxidizes a variety of substrates, including phenols and anilines, via the classic peroxidation cycle. The relative concentrations of chloride and the reducing substrate (AH) determine whether MPO uses hydrogen peroxide for chlorination or peroxidation. Assays based on measurement of chlorination activity are more specific for MPO than those based on peroxidase substrates such as tetramethylbenzidine (TMB).

The EnzChek Myeloperoxidase (MPO) Activity Assay Kit (E33856) provides assays for rapid and sensitive determination of both chlorination and peroxidation activities of MPO in solution and in cell lysates (Figure 18.24). For detection of chlorination, the kit provides nonfluorescent 3'-(p-aminophenyl) fluorescein (APF), which is selectively cleaved by hypochlorite (^–OCl) to yield fluorescein. Peroxidation is detected using nonfluorescent Amplex UltraRed reagent, which is oxidized by the H₂O₂-generated redox intermediates MPO-I and MPO-II to form a fluorescent product. The EnzChek Myeloperoxidase Activity Assay Kit can be used to continuously detect these activities at room temperature over a broad dynamic range (1.5 to 200 ng/mL) (Figure 18.25). The speed (30 minutes), sensitivity, and mix-and-read convenience make this kit ideal for measuring MPO activities and for high-throughput screening for MPO-specific inhibitors. Each EnzChek Myeloperoxidase (MPO) Activity Assay Kit contains:

• 3'-(p-aminophenyl) fluorescein (APF)
• Amplex UltraRed reagent
• Human myeloperoxidase (MPO) standard
• Chlorination inhibitor
• Peroxidation inhibitor
• Hydrogen peroxide (H₂O₂)
• Phosphate-buffered saline (PBS)
• Detailed protocols (EnzChek Myeloperoxidase (MPO) Activity Assay Kit)

Sufficient reagents are provided to perform 200 assays for chlorination and 200 assays for peroxidation activity in a 96-well fluorescence microplate format (100 µL per assay).

Zen Myeloperoxidase (MPO) ELISA Kit

The Zen Myeloperoxidase (MPO) ELISA Kit (Z33857) provides a comprehensive set of components for accurate and sensitive quantitation of MPO in a variety of biological samples, including human serum. This assay is based on Amplex UltraRed reagent, a fluorogenic substrate for horseradish peroxidase (HRP) that reacts with H₂O₂ in a 1:1 stoichiometric ratio to produce the brightly fluorescent and strongly absorbing Amplex UltraRed oxidation product (excitation/ emission maxima ~568/581 nm). Because the Amplex UltraRed product has long-wavelength emission, there is little interference from the blue or green autofluorescence found in most biological samples. With a high extinction coefficient, good quantum efficiency, and resistance to autooxidation, the fluorescence-based Amplex UltraRed reagent delivers better sensitivity and a broader assay range than colorimetric reagents. Each kit contains:

• Amplex UltraRed reagent
• Dimethylsulfoxide (DMSO)
• Concentrated phosphate-buffered saline (PBS)
• Horseradish peroxidase (HRP)–labeled goat anti–rabbit IgG antibody
• Amplex stop reagent
• Hydrogen peroxide (H₂O₂)
• MPO standard
• Bovine serum albumin (BSA)
• Tween 20
• Mouse anti-MPO antibody (primary capture antibody)
• Rabbit anti-MPO antibody (secondary capture antibody)
• Zen microplates
• Detailed protocols (Zen Myeloperoxidase (MPO) ELISA Kit)

Sufficient reagents are provided for 200 assays in a microplate format, using a 100 µL per well reaction volume. The Zen Myeloperoxidase (MPO) ELISA Kit can be used to detect from 0.2 to 100 ng/mL MPO at room temperature (Figure 18.26).

Figure 18.26 Typical standard curve for detection of MPO using the Zen Myeloperoxidase (MPO) ELISA Kit (Z33857). The sandwich ELISA was carried out as described in the protocol using a mouse anti-MPO primary capture antibody, MPO standards ranging from 0.2 ng/mL to 100 ng/mL, and a rabbit anti-MPO secondary capture antibody.

The generation of reactive oxygen species (ROS) is inevitable for aerobic organisms and, in healthy cells, occurs at a controlled rate. Under conditions of oxidative stress, however, ROS production is dramatically increased, resulting in subsequent alteration of membrane lipids, proteins and nucleic acids. Oxidative damage of these biomolecules is associated with aging and with a variety of pathological events, including atherosclerosis, carcinogenesis, ischemic reperfusion injury and neuorodegenerative disorders.

Assaying oxidative activity in live cells with fluorogenic, chemiluminescent or chromogenic probes is complicated by the frequent presence of multiple reactive oxygen species in the same cell. In addition, the nitric oxide radical (Probes for Nitric Oxide Research—Section 18.3) may produce the same changes in the optical properties of the probe as do other reactive oxygen molecules. Blocking agents and enzyme inhibitors can sometimes help to sort out the species responsible for the probe's optical response. Quantitative analysis can be further hindered due to: 1) the high intracellular concentration of glutathione, which can form thiyl or sulfinyl radicals or otherwise trap or reduce oxygen species; 2) the variable concentration of metals, which can either catalyze or inhibit radical reactions; and 3) the presence of other free radical–quenching agents such as spermine.

Fluorescein, rhodamine and various other dyes can be chemically reduced to colorless, nonfluorescent leuco dyes. These "dihydro" derivatives are readily oxidized back to the parent dye by reactive oxygen species and thus can serve as fluorogenic probes for detecting oxidative activity in cells and tissues; however, their oxidation may not easily discriminate between the various reactive oxygen species. It has been reported that dihydroethidium, dichlorodihydrofluorescein (H₂DCF) and dihydrorhodamine 123 react with intracellular hydrogen peroxide in a reaction mediated by peroxidase, cytochrome c or Fe²⁺, and these leuco dyes also serve as fluorogenic substrates for peroxidase enzymes (Substrates for Oxidases, Including Amplex Red Kits—Section 10.5). All of the nonfluorescence leuco dyes are slowly oxidized by air back to the parent fluorescent dyes, and in some cases light appears to accelerate their oxidation.

Dichlorodihydrofluorescein Diacetate

The cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA, D399; ), also known as dichlorofluorescin diacetate, is commonly used to detect the generation of reactive oxygen intermediates in neutrophils and macrophages. Upon cleavage of the acetate groups by intracellular esterases and oxidation, the nonfluorescent H₂DCFDA is converted to the highly fluorescent 2',7'-dichlorofluorescein (DCF). H₂DCFDA may also be extremely useful for assessing the overall oxidative stress in toxicological phenomena.

Oxidation of H₂DCFDA is reportedly not sensitive to singlet oxygen directly, but singlet oxygen can indirectly contribute to the formation of DCF through its reaction with cellular substrates that yield peroxy products and peroxyl radicals. In a cell-free system, H₂DCF has been shown to be oxidized to DCF by peroxynitrite anion (ONOO^–), by horseradish peroxidase (in the absence of H₂O₂) and by Fe²⁺ (in the absence of H₂O₂). Furthermore, the oxidation of H₂DCF by Fe²⁺ in the presence of H₂O₂ was reduced by the HO· radical scavenger formate and the iron chelator deferoxamine. In addition, DCF itself can act as a photosensitizer for H₂DCFDA oxidation, both priming and accelerating the formation of DCF. Because the oxidation of DCF and H₂DCFDA appears to also generate free radicals, their use for measuring free radical production must be carefully controlled.

A review by Tsuchiya and colleagues outlined methods for visualizing the generation of oxidative species in whole animals. For example, they suggest using propidium iodide (P1304MP; P3566; FluoroPure Grade—Note 19.2, P21493; Nucleic Acid Stains—Section 8.1) with H₂DCFDA to simultaneously monitor oxidant production and cell injury. H₂DCFDA has been used to visualize oxidative changes in carbon tetrachloride–perfused rat liver and in venular endothelium during neutrophil activation, as well as to examine the effect of ischemia and reperfusion in lung and heart tissue. Using H₂DCFDA, researchers characterized hypoxia-dependent peroxide production in Saccharomyces cerevisiae as a possible model for ischemic tissue destruction. A variety of toxicological phenomena in cultured cells have also been investigated with H₂DCFDA, including:

• Amyloid β protein–mediated increases in hydrogen peroxide in PC12 cells
• Effects of calcium antagonists on oxidative metabolism in dissociated rat cerebellar and cortical neurons
• Methamphetamine-induced oxidative stress in dopaminergic neurons
• Effect of lipopolysaccharides on the level of oxygen metabolites in rat liver Kupffer cells
• Nephrotoxin-induced oxidative stress in isolated proximal tubular cells
• Nickel-induced increases in oxidant levels in Chinese hamster ovary (CHO) cells
• Effect of transforming growth factor–β1 on the overall oxidized state of mouse osteoblastic cells

In neutrophils, H₂DCFDA has proven useful for flow cytometric analysis of nitric oxide, forming a product that has spectral properties identical to those produced when it reacts with hydrogen peroxide. In this study, H₂DCFDA's reaction with nitric oxide was blocked by adding the nitric oxide synthase inhibitor N^G-methyl-L-arginine (L-NMMA) to the cell suspension. 2',7'-Dichlorofluorescein—the oxidation product of H₂DCF—can reportedly be further oxidized to a phenoxyl radical in a horseradish peroxidase–catalyzed reaction, and this reaction may complicate the interpretation of results obtained with this probe in cells undergoing oxidative stress. H₂DCF is also photooxidized to fluorescent products by photoirradiation; in a cell-free system, this photoreaction can be suppressed by addition of ascorbic acid.

Improved Versions of H₂DCFDA

Intracellular oxidation of H₂DCF tends to be accompanied by leakage of the product, 2',7'-dichlorofluorescein, which may make quantitation or detection of slow oxidation difficult. To enhance retention of the fluorescent product, we offer the carboxylated H₂DCFDA analog (carboxy-H₂DCFDA, C400), which has two negative charges at physiological pH, and its di(acetoxymethyl ester) (C2938, , ), which should more easily pass through membranes during cell loading. Upon cleavage of the acetate and ester groups by intracellular esterases and oxidation , both analogs form carboxydichlorofluorescein (C368, Polar Tracers—Section 14.3), with additional negative charges that should impede its leakage out of the cell. Carboxy-H₂DCFDA (C400) has been used to assess the oxidative process in isolated perfused rat heart tissue and in transfected cos-1 cells expressing native or mutagenized prostaglandin endoperoxide H synthase. Its di(acetoxymethyl ester) (C2938) has been employed to investigate the role of the bcl-2 proto-oncogene product in preventing apoptosis through its antioxidant properties.

The fluorinated analog 5-(and 6-)carboxy-2',7'-difluorodihydrofluorescein diacetate (carboxy-H₂DFFDA, C13293) is also useful for studying oxidative bursts and reactive oxygen species. Carboxy-H₂DFFDA exhibits improved photostability when compared with other fluorescein derivatives in common use. The diacetate derivatives of the dichloro- and difluorodihydrofluoresceins are quite stable. When used for intracellular applications, the acetates are cleaved by endogenous esterases, releasing the corresponding dichloro- or difluorodihydrofluorescein derivative. If, however, these nonfluorescent diacetate derivatives are used for in vitro assays, they must first be hydrolyzed with mild base to form the colorless probe.

In addition, we have developed 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA, C6827; ; Figure 18.10), which is a chloromethyl derivative of H₂DCFDA that should exhibit much better retention in live cells. As with our other chloromethyl derivatives (see the description of our CellTracker probes in Membrane-Permeant Reactive Tracers—Section 14.2), CM-H₂DCFDA passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases and its thiol-reactive chloromethyl group reacts with intracellular glutathione and other thiols. Subsequent oxidation yields a fluorescent adduct that is trapped inside the cell, thus facilitating long-term studies. Among its many applications, CM-H₂DCFDA has been used to:

• Assay H₂O₂ diffusion through specific plant and mammalian aquaporins expressed in yeast
• Detect insulin-stimulated production of H₂O₂ in insulin-sensitive hepatoma and adipose cells
• Explore the parallel induction of reactive oxygen species and Ca²⁺ transients in ouabain-treated myocytes
• Measure intracellular reactive oxygen species in cardiac myocytes in human embryonic kidney 293 (HEK293) cells stably transfected with the human vanilloid receptor 1 (VR1) cation channel
• Monitor arsenic-induced production of oxyradicals by confocal laser-scanning microscopy

Figure 18.10 An oxidative burst was detected by flow cytometry of cells labeled with 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA, C6827). Jurkat cells were incubated with 100 nM CM-H2DCFDA. The cells were washed and resuspended in either phosphate-buffered saline (PBS, red) or PBS with 0.03% H2O2 (blue). The samples were analyzed on a flow cytometer equipped with a 488 nm argon-ion laser and a 525 ± 10 nm bandpass emission filter.

Image-iT LIVE Green Reactive Oxygen Species Detection Kit

The Image-iT LIVE Green Reactive Oxygen Species Detection Kit (I36007) provides the key reagents for detecting reactive oxygen species (ROS) in live cells, including:

• Carboxy-H₂DCFDA (5-(and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate)
• Hoechst 33342
• tert-Butyl hydroperoxide (TBHP)
• Dimethylsulfoxide (DMSO)
• Detailed protocols for fluorescence microscopy assays (Image-iT LIVE Green Reactive Oxygen Species Detection Kit)

This assay is based on carboxy-H₂DCFDA (5-(and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate), a reliable fluorogenic marker for ROS in live cells. In addition to carboxy-H₂DCFDA, this kit provides the common inducer of ROS production tert-butyl hydroperoxide (TBHP) as a positive control and the blue-fluorescent, cell-permeant nucleic acid stain Hoechst 33342. Oxidatively stressed and nonstressed cells can be effectively distinguished by fluorescence microscopy using this combination of dyes and the protocol provided.

Aminophenyl Fluorescein and Hydroxyphenyl Fluorescein

Developed by Nagano, 3'-(p-aminophenyl) fluorescein (APF, A36003) and 3'-(p-hydroxyphenyl) fluorescein (HPF, H36004) provide greater selectivity and stability than dichlorodihydrofluorescein diacetate (H₂DCFDA, also called dichlorofluorescin diacetate, D399) for ROS detection. H₂DCFDA is probably the most commonly used reagent for detecting intracellular ROS species despite its lack of specificity and tendency to spontaneously photooxidize. The nonfluorescent H₂DCFDA becomes fluorescent in the presence of a wide variety of ROS including, but not limited to, peroxyl (ROO·) and hydroxyl (HO·) radicals and the peroxynitrite anion (ONOO^–). In contrast, APF and HPF show much more limited reactivity and greater resistance to light-induced oxidation (Fluorescence response of APF, HPF and H2DCFDA to various reactive oxygen species (ROS)—Table 18.3). Both of these fluorescein derivatives are essentially nonfluorescent until they react with the hydroxyl radical or peroxynitrite anion (Figure 18.11). APF will also react with the hypochlorite anion (^–OCl), making it possible to use APF and HPF together to selectively detect hypochlorite anion (Detecting Chloride, Phosphate, Nitrite and Other Anions—Section 21.2). In the presence of these specific ROS, both APF and HPF yield a bright green-fluorescent product (excitation/emission maxima ~490/515 nm) and are compatible with all fluorescence instrumentation capable of visualizing fluorescein. Using APF, researchers have been able to detect the hypochlorite anion generated by activated neutrophils, a feat that has not been possible with traditional ROS indicators.

Dihydrocalcein AM

Calcein AM (C1430, C3099, C3100MP; Viability and Cytotoxicity Assay Reagents—Section 15.2) is extremely useful as a probe for the study of cell viability, adhesion, multidrug resistance, chemotaxis and other processes. We have combined the superior retention of calcein (the hydrolytic product of calcein AM in viable cells) and the oxidation sensitivity of the dihydrofluoresceins to yield the probe dihydrocalcein AM (D23805, ),provided specially packaged as a set of 20 vials, each containing 50 µg. Dihydrocalcein AM is freely permeant to cell membranes and is oxidized to green-fluorescent calcein, which has superior retention properties in cells that have intact membranes.

OxyBURST Green Reagents

Fc OxyBURST Green assay reagent (F2902) was developed in collaboration with Elizabeth Simons of Boston University to monitor the oxidative burst in phagocytic cells using fluorescence instrumentation. The Fc OxyBURST Green assay reagent comprises bovine serum albumin (BSA) that has been covalently linked to dichlorodihydrofluorescein (H₂DCF) and then complexed with purified rabbit polyclonal anti-BSA antibodies. When these immune complexes bind to Fc receptors, the nonfluorescent H₂DCF molecules are internalized within the phagovacuole and subsequently oxidized to green-fluorescent dichlorofluorescein (DCF); see Probes for Following Receptor Binding, Endocytosis and Exocytosis—Section 16.1 for a more complete description. Unlike dichlorodihydrofluorescein diacetate (H₂DCFDA), the Fc OxyBURST Green assay reagent does not require intracellular esterases for activation, making this reagent particularly suitable for detecting the oxidative burst in cells with low esterase activity such as monocytes. Fc OxyBURST Green assay reagent reportedly produces >8 times more fluorescence than does H₂DCFDA at 60 seconds and >20 times more at 15 minutes following internalization of the immune complex.

OxyBURST Green H₂HFF BSA (O13291) is a sensitive fluorogenic reagent for detecting extracellular release of oxidative products in a spectrofluorometer or a fluorescence microscope . This reagent comprises BSA that has been covalently linked to dihydro-2',4,5,6,7,7'-hexafluorofluorescein (H₂HFF), a reduced dye with improved stability. Unlike Fc OxyBURST Green assay reagent, OxyBURST Green H₂HFF BSA is not complexed with IgG. OxyBURST Green H₂HFF BSA provides up to 1000-fold greater sensitivity than conventional methods based on spectrophotometric detection of superoxide dismutase–inhibitable reduction of cytochrome c and allows researchers to take advantage of the sample stirring and temperature control capabilities available in many spectrofluorometers. Because OxyBURST Green H₂HFF BSA is a protein conjugate, it is superior to low molecular weight probes such as dihydrotetramethylrosamine and dihydrorhodamine 123, which are cell permeant and therefore do not exclusively detect extracellular oxidants.

Amine-Reactive OxyBURST Green Reagent

As an alternative to Fc OxyBURST Green assay reagent and OxyBURST Green H₂HFF BSA, we offer the amine-reactive OxyBURST Green H₂DCFDA succinimidyl ester (2',7'-dichlorodihydrofluorescein diacetate, SE; D2935; ), which can be used to prepare oxidation-sensitive conjugates of a wide variety of biomolecules and particles, including antibodies, antigens, peptides, proteins, dextrans, bacteria, yeast and polystyrene microspheres. Following conjugation to amines, the two acetates of OxyBURST Green H₂DCFDA can be removed by treatment with hydroxylamine at neutral pH to yield the dihydrofluorescein conjugate. OxyBURST Green H₂DCFDA conjugates are nonfluorescent until they are oxidized to the corresponding fluorescein derivatives. In one application, OxyBURST Green H₂DCFDA succinimidyl ester was conjugated to an antibody that binds specifically to YAC tumor cells. YAC cells opsonized with this customized OxyBURST reagent were then used in a fluorescence microscopy study to show that Fc receptor–activated neutrophils appear to deliver reactive oxygen species to the surface of their target cells.

Dihydrorhodamine 123

Dihydrorhodamine 123 (D632, D23806; , ) is the uncharged and nonfluorescent reduction product of the mitochondrion-selective dye rhodamine 123 (R302; FluoroPure Grade—Note 19.2, R22420; Probes for Mitochondria—Section 12.2). This leuco dye passively diffuses across most cell membranes where it is oxidized to cationic rhodamine 123 (), which localizes in the mitochondria. Like H₂DCF, dihydrorhodamine 123 does not directly detect superoxide, but rather reacts with hydrogen peroxide in the presence of peroxidase, cytochrome c or Fe²⁺. However, dihydrorhodamine 123 also reacts with peroxynitrite, the anion formed when nitric oxide reacts with superoxide. Peroxynitrite, which may play a role in many pathological conditions, has been shown to react with sulfhydryl groups, DNA and membrane phospholipids, as well as with tyrosine and other phenolic compounds.

Dihydrorhodamine 123 has been used to investigate reactive oxygen intermediates produced by human and murine phagocytes, activated rat mast cells and cultured endothelial cells. It has also been employed to study the role of the CD14 cell-surface marker in H₂O₂ production by human monocytes. In addition, dihydrorhodamine 123 has been used with the Fura Red calcium indicator (F3020, F3021; Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3) to simultaneously measure oxidative bursts and Ca²⁺ fluxes in monocytes and granulocytes. Dihydrorhodamine 123 is reportedly a more sensitive probe than H₂DCFDA for detecting granulocyte respiratory bursts.

Dihydrorhodamine 123 is available as a 10 mg vial (D632) or as a stabilized 5 mM solution in DMSO (D23806). Because of the susceptibility of dihydrorhodamine 123 to air oxidation, the DMSO solution is recommended when only small quantities are to be used at a time.

A Longer-Wavelength Reduced Rhodamine

Intracellular oxidation of dihydrorhodamine 6G (D633) yields rhodamine 6G (R634), which localizes in the mitochondria of live cells (Probes for Mitochondria—Section 12.2). As compared with rhodamine 123, this cationic oxidation product has longer-wavelength spectra, making it especially useful in multicolor applications and in autofluorescent cells and tissues. Dihydrorhodamine 6G has been used in the study of chloride conductance and mutations of the cystic fibrosis transmembrane conductance regulator.

Reduced MitoTracker Probes

Two of our MitoTracker probes—MitoTracker Orange CM-H₂TMRos (M7511, ) and MitoTracker Red CM-H₂XRos (M7513, )—are chemically reactive reduced rosamines. Unlike MitoTracker Orange CMTMRos and MitoTracker Red CMXRos (M7510, M7512; Probes for Mitochondria—Section 12.2), the reduced versions of these probes do not fluoresce until they enter an actively respiring cell, where they are oxidized by reactive oxygen species to the fluorescent mitochondrion-selective probe and then sequestered in the mitochondria. Both MitoTracker Orange CMTMRos and the reduced MitoTracker Orange CM-H₂TMRos have been used to investigate the metabolic state of Pneumocystis carinii mitochondria.

Dihydroethidium (Hydroethidine)

Although dihydroethidium (), which is also called hydroethidine, is commonly used to analyze respiratory burst in phagocytes, it has been reported that this probe undergoes significant oxidation in resting leukocytes, possibly through the uncoupling of mitochondrial oxidative phosphorylation. Cytosolic dihydroethidium exhibits blue fluorescence; however, once this probe is oxidized to ethidium (E1305, Nucleic Acid Stains—Section 8.1, it intercalates within DNA, staining the cell nucleus a bright fluorescent red (). The mechanism of dihydroethidium's interaction with lysosomes and DNA has been described. A recent report suggests that oxidation of dihydroethidium by superoxide produces a fluorescent product that is distinctly different from ethidium and that exhibits shorter-wavelength fluorescence upon binding to DNA. Dihydroethidium has been used for many purposes, including:

• Detecting multidrug-resistant cancer cells (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6)
• Following phagocytosis and oxidative bursts by phagocytic blood cells
• Investigating spermatozoal viability
• Quantitating killer cell–target cell conjugates by flow cytometry methods

Dihydroethidium (hydroethidine) is available in a 25 mg vial (D1168), as a stabilized 5 mM solution in DMSO (D23107) or specially packaged in 10 vials of 1 mg each (D11347); the stabilized DMSO solution or special packaging is recommended when small quantities of the dye will be used over a long period of time.

RedoxSensor Red CC-1 Stain

Localization of the RedoxSensor Red CC-1 stain (2,3,4,5,6-pentafluorotetramethyldihydrorosamine, R14060; ) appears to be based on a cell's cytosolic redox potential. Once it passively enters live cells, RedoxSensor Red CC-1 stain may be oxidized in the cytosol to a red-fluorescent product (excitation/emission maxima ~540/600 nm), which then accumulates in the mitochondria. Alternatively, this nonfluorescent probe may be transported to the lysosomes where it is oxidized. The differential distribution of the oxidized product between mitochondria and lysosomes appears to depend on the redox potential of the cytosol. In proliferating cells, mitochondrial staining predominates; whereas in contact-inhibited cells, the staining is primarily lysosomal (). The best method we have found to quantitate the distribution of the oxidized product is to use the mitochondrion-selective MitoTracker Green FM stain (M7514) in conjunction with the RedoxSensor Red CC-1 stain.

Glutathiolation Detection with BioGEE

Biotinylated glutathione ethyl ester (BioGEE, G36000; ) is a cell-permeant, biotinylated glutathione analog for the detection of glutathiolation. Under conditions of oxidative stress, cells may transiently incorporate glutathione into proteins. Stressed cells incubated with BioGEE will also incorporate this biotinylated glutathione derivative into proteins, facilitating the identification of oxidation-sensitive proteins. Once these cells are fixed and permeabilized, glutathiolation levels can be detected with a fluorescent streptavidin conjugate (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.23) using either flow cytometry or fluorescence microscopy. Proteins glutathiolated with BioGEE can also be extracted and analyzed by mass spectrometry or by Western blotting methods in conjunction with fluorophore- or enzyme-labeled streptavidin conjugates.

Tetrazolium salts—especially MTT (M6494, )—are widely used for detecting the redox potential of cells for viability, proliferation and cytotoxicity assays. Upon reduction, these water-soluble colorless compounds form uncharged, brightly colored formazans. Several of the formazans precipitate out of solution and are useful for histochemical localization of the site of reduction or, after solubilization in organic solvent, for quantitation by standard spectrophotometric techniques. The extremely water-soluble formazan product of XTT (X6493) does not require solubilization prior to quantitation.

Many of these salts are reduced by specific components of the electron transport chain and may be useful for determining the site of action of specific toxins. Selected applications of the tetrazolium salts are listed in Tetrazolium salts for detecting redox potential in living cells and tissues—Table 18.2. Our Vybrant MTT Cell Proliferation Assay Kit (V13154, Assays for Cell Enumeration, Cell Proliferation and Cell Cycle—Section 15.4) provides a means of counting metabolically active cells; this Vybrant MTT assay can detect from 2000 to 250,000 cells, depending on the cell type and conditions. See also Viability and Cytotoxicity Assay Reagents—Section 15.2 for additional cell applications of tetrazolium salts.