CHAPTER 1 CHAPTER 18 Fluorophores and Probes for Reactive ... · Free Radic Res (2000) 32:265; 66. Anal Biochem (1999) 271:53; 67. Free Radic Res Commun (1993) 18:369; 68. Arch Biochem

CHAPTER 18

Probes for Reactive Oxygen Species Including Nitric Oxide

Molecular Probestrade HandbookA Guide to Fluorescent Probes and Labeling Technologies

11th Edition (2010)

CHAPTER 1

Fluorophores and Their Amine-Reactive Derivatives

The Molecular Probesreg HandbookA GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES11th Edition (2010)

Molecular Probesreg Resources

Molecular Probesreg Handbook (online version)Comprehensive guide to uorescent probes and labeling technologies

lifetechnologiescomhandbook

Fluorescence SpectraViewerIdentify compatible sets of uorescent dyes and cell structure probes

lifetechnologiescomspectraviewer

BioProbesreg Journal of Cell Biology ApplicationsAward-winning magazine highlighting cell biology products and applications

lifetechnologiescombioprobes

Access all Molecular Probesreg educational resources at lifetechnologiescommpeducate

Molecular Probes ResourcesMolecular Probes Handbook (online version)Comprehensive guide to fl uorescent probes and labeling technologiesthermofi shercomhandbook

Molecular Probes Fluorescence SpectraViewerIdentify compatible sets of fl uorescent dyes and cell structure probesthermofi shercomspectraviewer

BioProbes Journal of Cell Biology ApplicationsAward-winning magazine highlighting cell biology products and applicationsthermofi shercombioprobes

Access all Molecular Probes educational resources at thermofi shercomprobes

803wwwinvitrogencomprobes

The Molecular Probesreg Handbook A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE The products described in this manual are covered by one or more Limited Use Label License(s) Please refer to the Appendix on page 971 and Master Product List on page 975 Products are For Research Use Only Not intended for any animal or human therapeutic or diagnostic use

EIG

HTE

EN

CHAPTER 18

Probes for Reactive Oxygen Species Including Nitric Oxide181 Introduction to Reactive Oxygen Species 805

182 Generating and Detecting Reactive Oxygen Species 806

Generating Singlet Oxygen 806

Hypericin 806

Rose Bengal Diacetate 806

Merocyanine 540 806

Detecting Singlet Oxygen 807

Singlet Oxygen Sensor Green Reagent 807

trans-1-(2acute-Methoxyvinyl)pyrene 807

Generating Hydroxyl and Superoxide Radicals 807

Malachite Green 807

110-Phenanthroline Iodoacetamide 807

Detecting Hydroxyl and Superoxide Radicals 808

MitoSOXtrade Red Mitochondrial Superoxide Indicator 808

Dihydroethidium (Hydroethidine) 808

Fluorogenic Spin Traps 809

Chemiluminescent and Chromogenic Reagents for Detecting Superoxide 809

Detecting Peroxides Peroxyl Radicals and Lipid Peroxidation 810

cis-Parinaric Acid 810

Diphenyl-1-Pyrenylphosphine 810

BODIPYreg 581591 C A Ratiometric Lipid Peroxidation Sensor 811

Other Scavengers for Peroxyl Radicals 811

Luminol 811

Detecting 4-Hydroxy-2-Nonenal 811

Detecting Peroxides and Peroxidases with Amplexreg Red Reagents 811

Amplexreg Red Reagent Stable Substrate for Peroxidase Detection 811

Amplexreg UltraRed Reagent Brighter and More Sensitive than the Amplexreg Red Reagent 812

Amplexreg Red Hydrogen PeroxidePeroxidase Assay Kit 812

Amplexreg Red XanthineXanthine Oxidase Assay Kit 812

EnzChekreg Myeloperoxidase (MPO) Activity Assay Kit 813

Zentrade Myeloperoxidase (MPO) ELISA Kit 814

Assaying Oxidative Activity in Live Cells 815

Dichlorodihydrouorescein Diacetate 815

Improved Versions of H2DCFDA 816

Image-iTreg LIVE Green Reactive Oxygen Species Detection Kit 817

Aminophenyl Fluorescein and Hydroxyphenyl Fluorescein 817

Dihydrocalcein AM 818

OxyBURSTreg Green Reagents 818

Amine-Reactive OxyBURSTreg Green Reagent 818

The Molecular Probestrade Handbook A Guide to Fluorescent Probes and Labeling Technologies

IMPORTANT NOTICE The products described in this manual are covered by one or more Limited Use Label License(s) Please refer to the Appendix on page 971 and Master Product List on page 975 Products are For Research Use Only Not intended for any animal or human therapeutic or diagnostic use

thermofi shercomprobes



Chapter 18 mdash Probes for Reactive Oxygen Species Including Nitric Oxide

Dihydrorhodamine 123 818

A Longer-Wavelength Reduced Rhodamine 819

Reduced MitoTrackerreg Probes 819

RedoxSensortrade Red CC-1 Stain 819

Glutathiolation Detection with BioGEE 819

Tetrazolium Salts Chromogenic Redox Indicators 820

Data Table 182 Generating and Detecting Reactive Oxygen Species 821

Product List 182 Generating and Detecting Reactive Oxygen Species 823

183 Probes for Nitric Oxide Research 824

Spontaneous Nitric Oxide Donors and Antagonist 824

Spermine NONOate 824

SNAP and SIN-1 824

Carboxy-PTIO A Nitric Oxide Antagonist 824

SNAP A Photoactivatable Nitric Oxide Donor 824

Detecting Nitric Oxide Nitrite and Nitrate 824

DAF-FM Nitric Oxide Indicator 824

23-Diaminonaphthalene 825

12-Diaminoanthraquinone 826

NBD Methylhydrazine 826

Dichlorodihydrouorescein Diacetate and Dihydrorhodamine 123 826

Anti-Nitrotyrosine Antibody 826

S-Nitrosothiol Detection 826

Griess Reagent Kit 827

Measure-iTtrade High-Sensitivity Nitrite Assay Kit 827

Data Table 183 Probes for Nitric Oxide Research 828

Product List 183 Probes for Nitric Oxide Research 828


IMPORTANT NOTICE The products described in this manual are covered by one or more Limited Use Label License(s) Please refer to the Appendix on page 971 and Master Product List on page 975 Products are For Research Use Only Not intended for any animal or human therapeutic or diagnostic usethermofishercomprobes




Section 181 Introduction to Reactive Oxygen Species

Activated oxygen species are produced during a number of physi-ological 1ndash3 and pathological 4ndash7 processes eir eects are through reactions with a large variety of easily oxidizable cellular components including NADH NADPH ascorbic acid histidine tryptophan ty-rosine cysteine glutathione proteins and nucleic acids8ndash12 Reactive oxygen species can also oxidize cholesterol and unsaturated fatty ac-ids causing membrane lipid peroxidation71314 Several reviews dis-cuss the chemistry of the dierent reactive oxygen species and their detection15ndash19

181 Introduction to Reactive Oxygen Species

Table 181 Reactive oxygen species

Reactive Oxygen Species Structure Detection Reagents

Hydrogen peroxide H2O2 bull Carboxy-H2DCFDA (C400) 1ndash3

bull CM-H2DCFDA (C6827) 45

bull Dihydrocalcein AM (D23805)bull Dihydrorhodamine 123 (D632 D23806) 6

bull Dihydrorhodamine 6G (D633) 7

bull H2DCFDA (D399) 8ndash11

bull Lucigenin (L6868) 1213

bull Luminol (L8455) 14

bull RedoxSensortrade Red CC-1 (R14060) 15

Hydroxyl radical HObull bull 3acute-(p-Aminophenyl) uorescein (APF A36003)bull 3acute-(p-Hydroxyphenyl) uorescein (HPF H36004)bull CM-H2DCFDA (C6827) 16

bull Proxyl uorescamine (C7924) 17

bull TEMPO-9-AC (A7923)

Hypochlorous acid HOCl bull Aminophenyl uorescein (APF A36003)bull Dihydrorhodamine 123 (D632 D23806) 18

bull Luminol (L8455) 19ndash21

Nitric oxide NO bull DAF-FM (D23841) 2223

bull DAF-FM diacetate (D23842 D23844) 2223

bull DAA (D23840) 24

bull 23-Diaminonaphthalene (D7918) 24


Peroxyl radical including both alkylperoxyl and hydroperoxyl 26 radicals (wherein R = H)

ROObull bull BODIPYreg FL EDA (D2390) 27

bull BODIPYreg 665676 (B3932) 28


bull Carboxy-H2DCFDA (C400) 34

bull CM-H2DCFDA (C6827)

bull DPPP (D7894) 35ndash37


bull cis-Parinaric acid (P36005) 4142


Peroxynitrite anion dagger ONOOndash bull 3acute-(p-Aminophenyl) uorescein (APF A36003)bull 3acute-(p-Hydroxyphenyl) uorescein (HPF H36004)bull H2DCFDA (D399) 4344

bull Carboxy-H2DCFDA (C400)bull CM-H2DCFDA (C6827)

bull Coelenterazine (C2944) 45

bull Dihydrorhodamine 123 (D632 D23806) 4346ndash48

bull Dihydrorhodamine 6G (D633)bull Luminol (L8455) 434950

Singlet oxygen Dagger 1O2 bull Singlet Oxygen Sensor Green reagent (S36002) bull trans-1-(2acute-methoxyvinyl)pyrene (M7913) 5152

Superoxide anion bullO2ndash bull Coelenterazine (C2944) 5354

bull Dihydroethidium (D1168 D11347 D23107) 5556

bull Fc OxyBURSTreg Green assay reagent (F2902) 5758

bull OxyBURSTreg Green H2DCFDA SE (D2935) 5960

bull OxyBURSTreg Green H2HFF BSA (O13291) 61



bull MCLA (M23800) 6566

bull MTT (M6494) 67

bull NBT (N6495) 68


bull TEMPO-9-AC (A7923)bull XTT (X6493) 69

Hydroxyl radicals can also be photosensitized by malachite green isothiocyanate (M689) or generated by a N-(110-phenanthrolin-5-yl)iodoacetamide (P6879) metalndashligand complex dagger 3-Nitrotyrosine a product of this potent nitrating reagent can be detected with an anti-nitrotyrosine antibody (A21285) Dagger Singlet oxygen can also be photosensitized by hypericin (H7476) rose bengal diacetate (R14000) and merocyanine 540 (M24571)1 Biol Pharm Bull (2000) 231153 2 J Neurosci (1999) 199209 3 J Biol Chem (1996) 27121505 4 J Biol Chem (2001) 27621938 5 Proc Natl Acad Sci U S A (1997) 9411557 6 Biochim Biophys Acta (1999) 1454275 7 Proc Natl Acad Sci U S A (2000) 978266 8 J Biol Chem (2001) 276514 9 J Immunol Methods (1989) 11753 10 Brain Res (1994) 635113 11 J Biol Chem (1999) 27437111 12 Analyst (1986) 3941 13 J Am Chem Soc (1979) 1015347 14 J Bone Miner Res (1992) 71139 15 Free Radic Biol Med (2000) 281266 16 Proc Natl Acad Sci U S A (2001) 981643 17 Anal Chem (1997) 694295 18 Nitric Oxide (1997) 1145 19 Biochim Biophys Acta (1991) 1097145 20 Luminescence (1999) 14239 21 Am J Physiol (1992) 263G719 22 Anal Biochem (2000) 287203 23 Angew Chem Int Ed Engl (1999) 383209 24 Neuroreport (1998) 94051 25 Anal Chem (1993) 651794 26 DNA Cell Biol (2002) 21251 27 J Biochem Biophys Methods (1997) 3523 28 J Agric Food Chem (2000) 481150 29 Toxicol Meth (1994) 4224 30 J Biol Chem (2000) 27540028 31 Anal Biochem (1983) 134111 32 Am J Physiol (1989) 257C347 33 Methods Enzymol (1984) 105352 34 J Biol Chem (1998) 2735294 35 J Chromatogr (1993) 62831 36 Anal Lett (1987) 20731 37 Methods Enzymol (1990) 186157 38 Free Radic Biol Med (1995) 181 39 Biomed Chromatogr (1990) 4131 40 Lipids (1998) 331235 41 J Biol Chem (1997) 27212328 42 Biochem Biophys Res Commun (1998) 244647 43 Free Radic Biol Med (2001) 30463 44 FEBS Lett (2000) 46889 45 Circ Res (1999) 841203 46 FASEB J 2001 47 Arch Biochem Biophys (2000) 373302 48 FASEB J (2000) 141061 49 J Biol Chem (1996) 27129223 50 Arch Biochem Biophys (1994) 310352 51 Biochem Biophys Res Commun (1984) 123869 52 Methods Enzymol (1986) 133569 53 Anal Biochem (1992) 206273 54 Free Radic Biol Med (2000) 29170 55 Circ Res (2001) 88824 56 J Biol Chem (2001) 27617621 57 J Leukoc Biol (1997) 62329 58 J Biol Chem (1995) 2708328 59 Immunology (1994) 83507 60 J Immunol Methods (1990) 130223 61 Biophys J (1998) 752577 62 Free Radic Biol Med (2000) 281232 63 J Biol Chem (1998) 2732015 64 J Immunol Methods (1992) 155151 65 Free Radic Res (2000) 32265 66 Anal Biochem (1999) 27153 67 Free Radic Res Commun (1993) 18369 68 Arch Biochem Biophys (1997) 342275 69 Plant Physiol (1998) 117491

Molecular Probesreg products include probes that either generate or detect various reactive oxygen species (Table 181) including singlet oxygen (1O2) superoxide anion (bullO2

ndash) hydroxyl radical (HObull) and various peroxides (ROORrsquo) and hydroperoxides (ROOH) Section 182 describes these probes and their applications in vitro and in vivo

e importance of the nitric oxide radical (abbreviated NO) and other reactive oxygen species as biological messengers is the focus of intense re-search20ndash22 Section 183 is devoted to our probes for promoting inhibiting or detecting nitric oxide production in a variety of experimental systems



thermofishercomprobes




Section 182 Generating and Detecting Reactive Oxygen Species

We oer an assortment of Molecular Probesreg products for the generation of reactive oxygen species (ROS) including singlet oxygen (1O2) superoxide (bullO2

ndash) hydroxyl radical (HObull) and various peroxides (ROORʹ) and hydroperoxides (ROOH) (Table 181) as well as for their uoro-metric detection in solution Although there are no sensors that reversibly 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 e 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

Generating Singlet OxygenSinglet oxygen is responsible for much of the physiological damage caused by reactive oxygen

species including nucleic acid modication through selective reaction with deoxyguanosine to form 8-hydroxydeoxyguanosine 12 (8-OHdG) e lifetime of singlet oxygen is suciently long (44 microseconds in water 3) to permit signicant diusion in cells and tissues4 In the labora-tory singlet oxygen is usually generated in one of three ways photochemically from dioxygen (3O2) using a photosensitizing dye (Figure 1821) chemically by thermal decomposition of a peroxide or dioxetane or by microwave discharge through an oxygen stream Singlet oxygen can be directly detected by its characteristic weak chemiluminescence at 1270 nm45

HypericinAmong the most ecient reagents for generating singlet oxygen is the photosensitizer hyper-

icin (H7476 Figure 1822) a natural pigment isolated from plants of the genus Hypericum is heat-stable dye exhibits a quantum yield for singlet oxygen generation in excess of 07 as well as high photostability making it an important agent for both anticancer and antiviral research67

Rose Bengal DiacetateRose bengal diacetate (R14000) is an ecient cell-permeant generator of singlet oxygen8ndash10

It is an iodinated xanthene derivative that has been chemically modied by the introduction of acetate groups (Figure 1823) ese modications inactivate both its uorescence and photosen-sitization 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 eective photosensitizer

Merocyanine 540Photoirradiation of merocyanine 540 (M24571) produces both singlet oxygen and other

reactive oxygen species including oxygen radicals11ndash14 Merocyanine 540 is oen used as a photo-sensitizer in photodynamic therapy15

182 Generating and Detecting Reactive Oxygen Species

1 Chem Res Toxicol (2010) 23568 2 Cytometry A (2009) 75475 3 J Biol Chem (2006) 28129011 4 Nat Clin Pract Cardiovasc Med (2008) 5811 5 J Neurosci (2009) 299090 6 Mol Cell (2009) 33627 7 Physiol Rev (2004) 841381 8 Mitochondrion (2007) 7106 9 Electrophoresis (2005) 262599 10 Angew Chem Int Ed Engl (2007) 46561 11 Br J Pharmacol (2004) 142231 12 Biochem Pharmacol (2003) 661527 13 J Biol Chem

REFERENCES(2008) 28315539 14 Chem Res Toxicol (2008) 21432 15 Methods Mol Biol (2010) 59457 16 Methods Cell Biol (2007) 80355 17 Free Radic Biol Med (2007) 43995 18 J Biochem Biophys Methods (2005) 6545 19 Am J Physiol Regul Integr Comp Physiol (2004) 286R431 20 Curr Opin Chem Biol (2010) 1443 21 Free Radic Biol Med (2009) 47684 22 Nitric Oxide (2009) 2192

Figure 1821 Fluorescence response and specic-ity of Singlet Oxygen Sensor Green reagent (S36002) to 1O2 A) Fluorescence measurements were made in a spec-trouorometer using excitationemission wavelengths of 488525 nm for solutions containing 1 microM Singlet Oxygen Sensor Green reagent and 10 microM methylene blue in 100 mM pH 75 Tris buer alone the singlet oxygen scavenger so-dium azide (NaN3 1 mM) or 50 D2O which increases the lifetime of 1O2 Measurements were made for 20-second pe-riods with 30-second intervals (indicated by grey bars) be-tween each measurement During the 30-second intervals the samples were exposed to laser radiation (630ndash680 nm lt5 mW) resulting in methylene bluendashphotosensitized gen-eration of 1O2 B) Fluorescence measurements were made in a spectrouorometer using excitationemission wave-lengths of 488525 nm for solutions of 50 mM pH 7 Tris buer with 1 mM xanthine containing either 1 microM Singlet Oxygen Sensor Green reagent or dihydrorhodamine 123 (D632 D23806) After ~20 seconds 50 mUmL of xanthine oxidase (XO) was added XO catalzyes the oxidation of xan-thine producing uric acid and superoxide Superoxide can spontaneously degrade to H2O2

0

Time (sec)

300100 200

2000

1000

0

Fluo

resc

ence

50 D2O

Tris buffer

NaN3

0

Time (sec)

300100 200

300

200

100

0

Fluo

resc

ence

Dihydrorhodamine 123

Singlet Oxygen Sensor Green

Addition of 50 mUmL xanthine oxidase

A

B







Figure 1822 Hypericin (H7476)

O

O

OH

OHOH

OHOH

OH

H3CH3C

Figure 1823 Rose bengal diacetate (R14000)

Detecting Singlet OxygenSinglet Oxygen Sensor Green Reagent

Unlike other uorescent 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 reactive oxygen species including hydroxyl radical (HObull) su-peroxide (bullO2

ndash) and nitric oxide (NO) (Figure 1821) Before reaction with singlet oxygen this probe initially exhibits weak blue uorescence 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 uorescence similar to that of uorescein (excitationemission maxima ~504525 nm)

We have observed that the uorescent product of Singlet Oxygen Sensor Green reagent can degrade with time in some solutions and that Singlet Oxygen Sensor Green reagent can become uorescent at alkaline pH in the absence of singlet oxygen Nevertheless with the proper controls the intensity of the green-uorescent signal can be correlated with singlet oxygen concentration without signicant interference from other reactive oxygen species e Singlet Oxygen Sensor Green reagent has demonstrated utility for detecting singlet oxygen in solution 1617 and in plant tissues1819

trans-1-(2acute-Methoxyvinyl)pyrenetrans-1-(2acute-Methoxyvinyl)pyrene (M7913) can be used to detect picomole quantities of sin-

glet oxygen in chemical and biological systems (Figure 1824) making this compound one of the most sensitive singlet oxygen probes currently available20ndash22 Furthermore this highly selective chemiluminescent probe does not react with other activated oxygen species such as hydroxyl radical superoxide or hydrogen peroxide

Generating Hydroxyl and Superoxide RadicalsHydroxyl and superoxide radicals have been implicated in a number of pathological condi-

tions including ischemia reperfusion and aging e superoxide anion (Table 181) may also play a role in regulating normal vascular function e hydroxyl radical is a very reactive oxygen species 23 that has a lifetime of about 2 nanoseconds in aqueous solution and a radius of diusion of about 20 Aring us it induces peroxidation only when it is generated in close proximity to its tar-get e hydroxyl radical can be derived from superoxide in a Fenton reaction catalyzed by Fe2+ or other transition metals as well as by the eect of ionizing radiation on dioxygen Superoxide is most eectively generated from a hypoxanthinendashxanthine oxidase generating system 24ndash26 (Figure 1821)

Malachite GreenMalachite green is a nonuorescent photosensitizer that absorbs at long wavelengths

(~630 nm) Its photosensitizing action can be targeted to particular cellular sites by conjugating malachite green isothiocyanate (M689 Figure 1825) to specic antibodies2728 Enzymes and other proteins within ~10 Aring of the binding site of the malachite greenndashlabeled 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 diusion from the site of their generation29

110-Phenanthroline IodoacetamideConjugation of the iodoacetamide of 110-phenanthroline (P6879 Figure 1826) to thiol-

containing ligands confers the metal-binding properties of this important complexing agent on the ligand For example the covalent copperndashphenanthroline complex of oligonucleotides or nucleic acidndashbinding molecules in combination with hydrogen peroxide acts as a chemical nuclease to selectively cleave DNA or RNA3031 Hydroxyl radicals or other reactive oxygen spe-cies appear to be involved in this cleavage3233

Figure 1824 Reaction of trans-1-(2acute-methoxyvinyl)pyrene (M7913) with singlet oxygen (1O2) yielding a dioxetane in-termediate that generates chemiluminescence (CL) upon decomposition to 1-pyrenecarboxaldehyde

C CH OCH3

H1O2

C

O O

CH H

OCH3

CL (Em = 465 nm)

CH

O

Figure 1825 Malachite green isothiocyanate (M689)

C

N(CH3)2(CH3)2N

N C S

ClO4

Figure 1826 N-(110-phenanthrolin-5-yl)iodoacetamide (P6879)

N N

NH C CH2I

O








Detecting Hydroxyl and Superoxide RadicalsMitoSOXtrade Red Mitochondrial Superoxide Indicator

Mitochondrial superoxide is generated as a by-product of oxida-tive phosphorylation In an otherwise tightly coupled electron trans-port chain approximately 1ndash3 of mitochondrial oxygen consumed is incompletely reduced these leaky electrons can quickly interact with molecular oxygen to form superoxide anion the predominant reactive oxygen species in mitochondria3435 Increases in cellular su-peroxide production have been implicated in cardiovascular diseas-es including hypertension atherosclerosis and diabetes-associated vascular injuries36 as well as in neurodegenerative diseases such as Parkinson disease Alzheimer disease and amyotrophic lateral scle-rosis (ALS)35

MitoSOXtrade Red mitochondrial superoxide indicator (M36008) is a cationic derivative of dihydroethidum (also known as hydroethidine see below) designed for highly selective detection of superoxide in the mitochondria of live cells (Figure 1827) e cationic triphenylphos-phonium substituent of MitoSOXtrade Red indicator is responsible for the electrophoretically driven uptake of the probe in actively respir-ing mitochondria Oxidation of MitoSOXtrade Red indicator (or dihy-droethidium) by superoxide results in hydroxylation at the 2-position (Figure 1828) 2-hydroxyethidium (and the corresponding derivative of MitoSOXtrade Red indicator) exhibits a uorescence excitation peak at ~400 nm 37 that is absent in the excitation spectrum of the ethidium oxidation product generated by reactive oxygen species other than su-peroxide us uorescence excitation at 400 nm with emission detec-tion at ~590 nm provides optimum discrimination of superoxide from other reactive oxygen species 37ndash39 (Figure 1829)

Measurements of mitochondrial superoxide generation us-ing MitoSOXtrade Red indicator in mouse cortical neurons expressing caspase-cleaved tau microtubule-associated protein have been cor-related with readouts from uorescent indicators of cytosolic and mi-tochondrial calcium and mitochondrial membrane potential40 e relationship of mitochondrial superoxide generation to dopamine transporter activity measured using the aminostyryl dye substrate 4-Di-1-ASP (D288 Section 122) has been investigated in mouse brain astrocytes41 MitoSOXtrade Red indicator has been used for con-focal microscopy analysis of reactive oxygen species (ROS) produc-tion by mitochondrial NO synthase (mtNOS) in permeabilized cat ventricular myocytes 42 and in combination with Amplexreg Red re-agent for measurement of mitochondrial superoxide and hydrogen peroxide production in rat vascular endothelial cells43 In addition to imaging and microscope photometry measurements several ow cytometry applications of MitoSOXtrade Red indicator have also been reported Detailed protocols for simultaneous measurements of mito-chondrial superoxide generation and apoptotic markers APC annexin V (A35110 Section 155) and SYTOXreg Green (S7020 Section 81) in human coronary artery endothelial cells by ow cytometry have been published by Mukhopadhyay and co-workers44

Dihydroethidium (Hydroethidine)Although dihydroethidium (Figure 18210) which is also called

hydroethidine is commonly used to analyze respiratory bursts in phagocytes45 it has been reported that this probe undergoes signicant

Figure 1827 Detection of superoxide in live cells using MitoSOXtrade Red superoxide indica-tor (M36008) Live 3T3 cells were treated with FeTCPP a superoxide scavenger (right) or left untreated (left) Cells were then labeled with MitoSOXtrade Red reagent which uoresces when oxidized by superoxide and nuclei were stained with blue-uorescent Hoechst 33342 The mitochondria of untreated cells exhibited red uorescence indicating the presence of super-oxide whereas the mitochondria of treated cells showed minimal uorescence

Figure 18210 Dihydroethidium (hydroethidine D1168)

H H

HCHCH3

Figure 1828 Oxidation of MitoSOXtrade Red mitochondrial superoxide indicator to 2-hydroxy-5-(triphenylphosphonium)hexylethidium by superoxide (bullO2

ndash)

N

(CH2)6 P

H2N NH2

H

3

N

(CH2)6 P

H2N NH2

3

OH

O2

Figure 1829 Selectivity of the MitoSOXtrade Red mitochondrial superoxide indicator (M36008) Cell-free systems were used to generate a variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS) each oxidant was then added to a separate 10 microM solu-tion of MitoSOXtrade Red reagent and incubated at 37degC for 10 minutes Excess DNA was add-ed (unless otherwise noted) and the samples were incubated for an additional 15 minutes at 37degC before uorescence was measured The Griess Reagent Kit (G7921) (for nitric oxide peroxynitrite and nitrite standards only blue bars) and dihydrorhodamine 123 (DHR 123 (D632) green bars) were employed as positive controls for oxidant generation Superoxide dismutase (SOD) a superoxide scavenger was used as a negative control for superoxide The results show that the MitoSOXtrade Red probe (red bars) is readily oxidized by superoxide but not by the other oxidants

1

10

100

1000

10000

Super

oxide

Super

oxide +

SOD

No ad

ditionH 2

O 2

H 2O 2

+ H

RP

Singlet

oxy

gen

Perox

ynitr

ite

Super

oxide

Super

oxide +

SOD

Super

oxide (

no D

NA)

No ad

dition

H 2O 2

H 2O 2

+ H

RP

Nitric

oxide

Singlet

oxy

gen

Perox

ynitr

ite

Rel

ativ

e u

ores

cenc

e

0

02

04

06

08

10

12

14

16

Sodiumnitrite

Nitricoxide

Peroxy-nitrite

Ab

sorb

ance

(548

nm

)







oxidation in resting leukocytes possibly through the uncoupling of mitochondrial oxidative phosphorylation46 Cytosolic dihydroethidium exhibits blue uorescence however once this probe is oxidized to ethidium it intercalates within DNA staining the cell nucleus a bright uo-rescent red 47ndash49 (Figure 18211) e mechanism of dihydroethidiumrsquos interaction with lyso-somes and DNA has been described50 Similar to MitoSOXtrade Red mitochondrial superoxide in-dicator (Figure 1828) dihydroethidium is oxidized by superoxide to 2-hydroxyethidium37 It is frequently used for mitochondrial superoxide detection51ndash54 although MitoSOXtrade Red indicator provides more specic mitochondrial localization Indeed in some cases researchers have used dihydroethidium and MitoSOXtrade Red indicator to provide discrete indications of cytosolic and mitochondrial superoxide production respectively55

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 stabi-lized DMSO solution or special packaging is recommended when small quantities of the dye will be used over a long period of time

Fluorogenic Spin TrapsHydroxyl radicals have usually been detected aer reaction with spin traps We oer

TEMPO-9-AC (A7923 Figure 18212) and proxyl uorescamine 56ndash59 (C7924 Figure 18213) two uorogenic probes for detecting hydroxyl radicals 60 and superoxide Each of these molecules contains a nitroxide moiety that eectively quenches its uorescence However once TEMPO-9-AC or proxyl uorescamine traps a hydroxyl radical or superoxide its uorescence is restored and the radicalrsquos electron spin resonance signal is destroyed making these probes useful for detecting radicals either by uorescence or by electron spin resonance spectroscopy TEMPO-9-AC has been reported to detect glutathionyl radicals but not phenoxyl radicals61 Proxyl uores-camine can be used to detect the methyl radicals that are formed by reacting hydroxyl radicals with DMSO59 Radical-specic scavengers (Table 182)mdashsuch as the superoxide-specic p-ben-zoquinone and superoxide dismutase 62 or the hydroxyl radicalndashspecic mannitol and dimethyl-sulfoxide (DMSO) 566364mdashcan be used to identify the detected species

Chemiluminescent and Chromogenic Reagents for Detecting SuperoxideIn the absence of apoaequorin the luminophore coelenterazine (C2944) produces chemi-

luminescence in response to superoxide generation in cells organelles bacteria 65 and tissues66 Unlike luminol coelenterazine exhibits luminescence that does not depend on the activity of cell-derived myeloperoxidase and is not inhibited by azide67

In addition to coelenterazine we oer MCLA (M23800 Figure 18214) for detecting su-peroxide68 MCLA and coelenterazine are superior alternatives to lucigenin 65 (L6868) for this application because lucigenin can reportedly sensitize superoxide production leading to false-positive results69ndash73 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 lucigenin74

Table 182 Scavengers of reactive oxygen species (ROS)

ROS Scavenger (Working Concentration) References

Hydrogen peroxide (H2O2) Sodium pyruvate (10 mM) DMTU (10 mM) 1

Hydroxyl radical (HObull) Mannitol (20ndash100 mM) DMSO (028 M) dagger 1

Nitric oxide (NO) Carboxy-PTIO (C7912 100 microM) 1

Peroxyl radical (ROObull) Trolox Dagger (10ndash100 microM) α-tocopherol (10ndash100 microM) 23

Peroxynitrite anion (ONOOndash) Ebselen sect (10ndash100 microM) uric acid (100 microM) 14

Singlet oxygen (1O2) Sodium azide (1ndash10 mM) 1

Superoxide anion (bullO2ndash) MnTBAP (100 microM) Tiron daggerdagger (10 mM) 56

DMTU = NNʹ-dimethylthiourea Disproportionation by catalase is also widely used for suppression of H2O2 dagger 028 M DMSO = 2 (vv) The reactivity of HObull is so high that it can be argued that the actions of these reagents must be indirect Dagger Trolox = 6-hydroxy-2578-tetramethylchroman-2-carboxylic acid sect Ebselen = 2-phenyl-12-benzisoselenazol-3(2H)-one MnTBAP = manganese(III)-tetrakis(4-benzoic acid)porphyrin daggerdagger Tiron = 45-dihydroxybenzene-13-disulfonate1 J Biol Chem (2007) 28230452 2 Nitric Oxide (2006) 15163 3 Biochim Biophys Acta (2004) 1636136 4 J Biol Chem (2004) 2794425 5 Circ Res (2004) 9437 6 Bioorg Med Chem (2002) 103013

Figure 18211 Live bovine pulmonary artery endothelial cells (BPAEC) were incubated with the cell-permeant weakly blue-uorescent dihydroethidium (D1168 D11347 D23107) and the green-uorescent mitochondrial stain MitoTrackerreg Green FMreg (M7514) Upon oxidation red-uorescent ethid-ium accumulated in the nucleus

Figure 18212 4-((9-acridinecarbonyl)amino)-2266-tet-ramethylpiperidin-1-oxyl free radical (TEMPO-9-AC A7923)

N

C

NH

O

N

O

CH3

CH3

H3C

H3C

Figure 18213 5-(2-carboxyphenyl)-5-hydroxy-1-((2255-tetramethyl-1-oxypyrrolidin-3-yl)methyl)-3-phenyl-2-pyrro-lin-4-one potassium salt (proxyl uorescamine C7924)

N

O

CH3

CH3

H3C

H3C

NCH2

OHO C

O

KO

Figure 18214 2-methyl-6-(4-methoxyphenyl)-37-dihydro-imidazo12-apyrazin-3-one hydrochloride (MCLA M23800)

CH3OH

OCH3 HC








Nitro blue tetrazolium salt (NBT N6495 Table 183) and other tetrazolium salts are chromogenic probes useful for superoxide deter-mination7576 e superoxide sensitivity of tetrazolium salts can be a confounding factor in their more common applications for cell viability and proliferation assays77

Detecting Peroxides Peroxyl Radicals and Lipid Peroxidation

In peroxisomes H2O2 is produced by several enzymes that use mo-lecular oxygen to oxidize organic compounds is 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 bloodstream78ndash81 However H2O2 also plays a role in neurodegenerative and other disorders through induction of apoptosis 82 and DNA strand breaks83 modication of intracellular Ca2+ levels and mitochondrial potential and oxidation of glutathione In addition H2O2 is released from cells during hypoxia84

Peroxidation of unsaturated lipids aects cell membrane proper-ties85 signal transduction pathways8687 apoptosis and the deteriora-tion of foods and other biological compounds88 Lipid hydroperoxides have been reported to accumulate in oxidatively stressed individuals including HIV-infected patients89 Lipid peroxidation may also be re-sponsible for aging as well as for pathological processes such as drug-induced phototoxicity and atherosclerosis90 and is oen the cause of

free radicalndashmediated 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 91ndash93 (eg 4-hydroxy-2-nonenal or malonaldehyde see below)

Peroxyl radicals are formed by the decomposition of various peroxides and hydroperoxides including lipid hydroperoxides e hydroperoxyl radical is also the protonated form of superoxide and approximately 03 of the superoxide in the cytosol is present as this protonated radical94 Experimentally peroxyl radicals including alkyl-peroxyl (ROObull) and hydroperoxyl (HOObull) radicals are generated from compounds such as 22acute-azobis(2-amidinopropane) and from hydro-peroxides such as cumene hydroperoxide

cis-Parinaric AcidFluorescence quenching of the fatty acid analog cis-parinaric acid

(P36005) has been used in several lipid peroxidation assays95ndash97 in-cluding quantitative determinations in live cells9899 Parinaric acidrsquos extensive unsaturation (Figure 18215) makes it quite susceptible to oxidation if not rigorously protected from air100 Consequently we of-fer cis-parinaric acid in a 10 mL unit size of a 3 mM solution in de-oxygenated ethanol (P36005) if stored protected from light under an inert argon atmosphere at ndash20degC this stock solution should be stable for at least 6 months During experiments we advise handling parinaric acid samples under inert gas and preparing solutions using degassed buers and solvents Parinaric acid is also somewhat photolabile and undergoes photodimerization when exposed to intense illumination resulting in loss of uorescence101

Diphenyl-1-PyrenylphosphineHydroperoxides in lipids serum tissues and foodstus can be di-

rectly detected using the uorogenic reagent diphenyl-1-pyrenylphos-phine 102103 (DPPP D7894) DPPP is essentially nonuorescent until oxidized to a phosphine oxide by peroxides in vitro DPPP remains nonuorescent in the presence of hydroxyl radicals generated by the Cu2+-ascorbate method104 DPPP has previously been used to detect

Table 183 Tetrazolium salts for detecting redox potential in living cells and tissues

Cat No Tetrazolium SaltColor of Formazan

Water Solubility of Formazan Applications

M6494 (MTT) 3-(45-Dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide

purple no bull Superoxide generation by fumarate reductase 1 and nitric oxide synthase 2

bull Mitochondrial dehydrogenase activity 3

bull Cell viability and proliferation 4ndash9

bull Neuronal cell death 10

bull Platelet activation 11

bull Tumor cell adhesion 12 and invasion 13

bull Multidrug resistance 14

bull In vitro toxicity testing 15ndash17

N6495 (NBT) Nitro blue tetrazolium chloride deep blue no bull Superoxide generation by xanthine oxidase 18

bull Neutrophil oxidative metabolism 1920

bull NADPH diaphorase activity 21ndash23

bull Succinic dehydrogenase histochemistry 24

X6493 (XTT) 23-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide

orange yes bull Antifungal susceptibility 25

bull Drug sensitivity of cells 26

bull Parasitic nematode viability 27

bull Tumor cell cytotoxicity 28

1 J Biol Chem (1995) 27019767 2 J Biol Chem (1994) 26912589 3 Cytometry (1992) 13532 4 Biotechniques (1998) 25622 626 5 J Immunol Methods (1994) 168253 6 Anal Biochem (1993) 214190 7 J Immunol Methods (1993) 164149 8 Anal Biochem (1992) 2058 9 J Immunol Methods (1986) 89271 10 J Cell Biol (1995) 128201 11 J Immunol Methods (1993) 159253 12 J Immunol Methods (1993) 164255 13 Cancer Res (1994) 543620 14 Leuk Res (1992) 161165 15 Biosci Biotechnol Biochem (1992) 561472 16 J Immunol Methods (1991) 144141 17 J Immunol Methods (1990) 131165 18 J Reprod Fertil (1993) 97441 19 Clin Chim Acta (1993) 221197 20 J Leukoc Biol (1993) 53404 21 Neurosci Lett (1993) 15561 22 Proc Natl Acad Sci U S A (1991) 887797 23 Proc Natl Acad Sci U S A (1991) 882811 24 Histochemistry (1982) 76381 25 Antimicrob Agents Chemother (1992) 361619 26 Cancer Res (1988) 484827 27 Parasitology (1993) 107175 28 J Immunol Methods (1992) 147153

Figure 18215 cis-parinaric acid (P36005)

(CH2)7CCCC

CCCC

H

CH3CH2

H H

H

H

H

H

H

C OH

O







Figure 18216 44-diuoro-5-(4-phenyl-13-butadienyl)-4-bora-3a4a-diaza-s-indacene-3-undecanoic acid (BODIPYreg 581591 C11 D3861)

Figure 18217 Amplexreg Red reagent (A12222)

N

O OHHO

C CH3

O

Figure 18218 Principle of coupled enzymatic assays using Amplexreg Red reagent Oxidation of glucose by glucose oxidase results in generation of H2O2 which is coupled to conversion of the Amplexreg Red reagent to uorescent resorun by HRP The detection scheme shown here is used in the Amplexreg Red GlucoseGlucose Oxidase Assay Kit (A22189)

Amplexreg Red ResorunPeroxidase

Glucose Oxidase

H2O2 O2

N

O OHHO

C CH3O

N

O OHO

OHO

HO

CH2OH

O

OH

OHO

HO

CH2OHOH

OH

pico mole levels of hydroperoxides by HPLC105106 Its solubility in lipids makes DPPP quite useful for detecting hydroperoxides in the mem-branes of live cells 104107108 and in low-density lipoprotein particles109

BODIPYreg 581591 C A Ratiometric Lipid Peroxidation Sensor

e BODIPYreg 581591 C11 fatty acid (D3861 Figure 18216) is a sensitive uorescent reporter for lipid peroxidation undergoing a shi from red to green uorescence emission upon oxidation of the phen-ylbutadiene segment of the uorophore110 is oxidation-dependent emission shi enables uorescence ratio imaging of lipid peroxidation in live cells111ndash113 Other common applications of BODIPYreg 581591 C11 include uorometric assays of antioxidant ecacy in plasma 114115 and in lipid vesicles116 e oxidation and nitroxidation products of this BODIPYreg fatty acid have been characterized by mass spectrom-etry117118 Based on mass spectrometry analysis of oxidation products MacDonald and co-workers report that BODIPYreg 581591 C11 is more sensitive to oxidation than endogenous lipids and therefore tends to overestimate oxidative damage and underestimate antioxidant protec-tion eects119

Peroxyl radicals have also been detected in erythrocyte and red blood cell membranes using BODIPYreg FL EDA 120 (D2390 Section 34) a water-soluble BODIPYreg dye or BODIPYreg FL hexadecanoic acid (D3821 Section 132) BODIPYreg FL hexadecanoic acid exhibits the red shi common to the uorescence of lipophilic BODIPYreg dyes when they are concentrated permitting ratiometric measurements of hydroxyl radical production and allowing the onset of lipid peroxidation in live cells to be monitored121

Other Scavengers for Peroxyl Radicalse uorescence of several other probes is lost following interac-

tion with peroxyl radicals Lipophilic uorescein dyes such as hexa-decanoylaminouorescein 122 (H110 Section 135) and uorescein-labeled phosphatidylethanolamine (F362 Section 132) 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 Section 64) and pheno-lic dyes such as uorescein (F1300 F36915 Section 101) are extensively used as substrates in total antioxidant capacity assays of plasma and foods115123124

LuminolAlthough luminol (L8455) is not useful for detecting superoxide

in live cells125 it is commonly employed to detect peroxidase- or metal ionndashmediated oxidative events126ndash128 Used alone luminol can detect ox-idative events in cells rich in peroxidases including granulocytes 129ndash132 and spermatozoa133 is probe has also been used in conjunction with

horseradish peroxidase (HRP) to investigate reoxygenation injury in rat hepatocytes134135 In these experiments it is thought that the primary species being detected is hydrogen peroxide In addition luminol has been employed to detect peroxynitrite generated from the reaction of nitric oxide and superoxide136ndash138

Detecting 4-Hydroxy-2-NonenalFormation of 4-hydroxy-2-nonenal (HNE) from linoleic acid is a

major cause of lipid peroxidationndashinduced toxicity Several reagents for the direct uorometric detection of aldehydes are described in Section 33 e biotinylated hydroxylamine ARP (A10550 Section 42) is particularly useful for this purpose139 Biotinylation using click chemistry coupling (Section 31) enables anity purication of HNE-modied proteins140

Detecting Peroxides and Peroxidases with Amplexreg Red ReagentsAmplexreg Red Reagent Stable Substrate for Peroxidase Detection

In the presence of horseradish peroxidase (HRP) Amplexreg Red reagent (10-acetyl-37-dihydroxyphenoxazine A12222 A22177 Figure 18217) reacts with H2O2 in a 11 stoichiometry to produce highly uorescent resorun 141 (R363 Section 101 Figure 18218) Amplexreg Red reagent has greater stability yields less background and produces a red-uorescent 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 va-riety of membrane systems142ndash144








Amplexreg Red reagent has been used to detect the release of H2O2 from activated human leukocytes141145 to measure the activity of monoamine oxidase in bovine brain tissue146 to demonstrate the extracellular production of H2O2 produced by UV light stimulation of human keratinocytes 147ndash149 and for microplate assays of H2O2 and lipid hydroperoxide generation by isolated mitochondria5168150 Amplexreg 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)

Amplexreg UltraRed Reagent Brighter and More Sensitive than the Amplexreg Red Reagent

Amplexreg UltraRed reagent (A36006) improves upon the performance of Amplexreg Red re-agent oering brighter uorescence and enhanced sensitivity on a per-mole basis in horseradish peroxidase or horseradish peroxidasendashcoupled enzyme assays (Figure 18219) Fluorescence of oxidized Amplexreg UltraRed reagent is also less sensitive to pH (Figure 18220) and the substrate and its oxidation product exhibit greater stability than Amplexreg Red reagent in the presence of H2O2 or thiols such as dithiothreitol (DTT) Like Amplexreg Red reagent nonuorescent Amplexreg UltraRed reagent reacts with H2O2 in a 11 stoichiometric ratio to produce a brightly uorescent and strongly absorbing reaction product (excitationemission maxima ~568581 nm) (Figure 18221) Although the primary applications of the Amplexreg UltraRed reagent are enzyme-linked immunosorbent assays (ELISAs see Zentrade Myeloperoxidase ELISA Kit below) and in vitro anti-oxidant capacity assays151 it is also frequently used (in combination with HRP) to detect H2O2 production by isolated mitochondria 152 and cell cultures153154

Amplexreg Red Hydrogen PeroxidePeroxidase Assay Kite Amplexreg Red Hydrogen PeroxidePeroxidase Assay Kit (A22188) provides a simple

sensitive one-step assay for detecting H2O2 or the activity of horseradish peroxidase either by measuring uorescence with a uorescence-based microplate reader or a uorometer (Figure 18222) or by measuring absorption with an absorption-based microplate reader or a spectrophotometer e Amplexreg Red peroxidase substrate can detect the presence of ac-tive peroxidases and the release of H2O2 from biological samples including cells and cell extracts51141155156

e Amplexreg Red Hydrogen PeroxidePeroxidase Assay Kit contains

bull Amplexreg Red reagentbull Dimethylsulfoxide (DMSO)bull Horseradish (HRP)bull H2O2 for use as a positive controlbull Concentrated reaction buerbull Detailed protocols

Each kit provides sucient reagents for approximately 500 assays using a uorescence- or absorption-based microplate reader and a reaction volume of 100 microL per assay Several additional kits that utilize the Amplexreg Red peroxidase substrate to detect H2O2 in coupled enzymatic reac-tions are described in Section 105

Amplexreg Red XanthineXanthine Oxidase Assay KitXanthine oxidase (EC 1232) plays a key role in the production of free radicals in-

cluding superoxide in the body e Amplexreg Red XanthineXanthine 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 reac-tion mixture the superoxide spontaneously degrades to H2O2 which in the presence of HRP reacts stoichiometrically with Amplexreg Red reagent to generate the red-uorescent oxida-tion product resorun Resorun has absorption and uorescence emission maxima of ap-proximately 571 nm and 585 nm (Figure 18223) respectively and because the extinction coecient is high (54000 cmndash1Mndash1) the assay can be performed either uorometrically or spectrophotometrically

Figure 18219 Detection of H2O2 using Amplexreg UltraRed reagent (red squares) or Amplexreg Red reagent (blue tri-angles) Reactions containing 50 microM Amplexreg UltraRed or Amplexreg Red reagent 1 UmL HRP and the indicated amount of H2O2 in 50 mM sodium phosphate buer pH 74 were incubated for 30 minutes at room temperature The inset shows the sensitivity and linearity of the Amplexreg UltraRed assay at low levels of H2O2

30201000

100

500

400

300

200

0 200 400 600 800 1000

Hydrogen peroxide (nM)

0

10000

5000

15000

20000

Fluo

resc

ence

Amplexreg

Red

Amplexreg

UltraRed

Figure 18220 Comparison of pH-dependent uorescence of the products derived from oxidation of Amplexreg UltraRed reagent (solid blue circles) and Amplexreg Red reagent (open blue squares) Fluorescence intensities were measured using excitationemission of ~570585 nm

1 2 3 4 5 6 7 8 9 10

pH

Fluo

resc

ence

em

issi

on

Amplexreg Red

Amplexreg UltraRed

Figure 18221 Absorption and uorescence emission spectra of the product generated by horseradish peroxidasendashmediated oxidation of the Amplexreg UltraRed reagent in pH 75 buer

Wavelength (nm)

Ab

sorp

tion

Fluo

resc

ence

em

issi

on

400 650600450 500 550 700







Figure 18222 Detection of HRP using the Amplexreg Red Hydrogen PeroxidePeroxidase Assay Kit (A22188) Reactions con-taining 50 microM Amplexreg Red reagent 1 mM H2O2 and the indicated amount of HRP in 50 mM sodium phosphate buer pH 74 were incubated for 30 minutes at room temperature Fluorescence was measured with a uorescence microplate reader using excitation at 530 plusmn 125 nm and uorescence detection at 590 plusmn 175 nm Background uorescence (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

HRP (mUmL)

Fluo

resc

ence

00

500

1000

1500

2000

2500

3000

3500

1 2 3 4 5 6 7

0

100

200

300

400

500

0 01 02 03 04

Each kit provides sucient reagents for approximately 400 assays using either a uores-cence- or absorption-based microplate reader and a reaction volume of 100 microL 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 circula-tion erefore determination of serum xanthine oxidase levels serves as a sensitive indicator of acute liver damage such as jaundice e Amplexreg Red xanthinexanthine oxidase assay has been used as a marker of recovery from exercise stress157 Previously researchers have utilized chemi-luminescence or absorbance to monitor xanthine oxidase activity e Amplexreg Red XanthineXanthine Oxidase Assay Kit permits the detection of xanthine oxidase in a puried system at levels as low as 01 mUmL by uorescence (Figure 18224) is kit can also be used to detect as little as 200 nM hypoxanthine or xanthine (Figure 18225) and when coupled to the purine nucleotide phosphorylase enzyme to detect inorganic phosphate158

EnzChekreg Myeloperoxidase (MPO) Activity Assay KitMyeloperoxidase (MPO EC 11117) is a lysosomal hemoprotein located in the azurophilic

granules of polymorphonuclear (PMN) leukocytes and monocytes It is a dimeric protein com-posed of two 59 kD and two 135 kD subunits MPO is a unique peroxidase that catalyzes the conversion of hydrogen peroxide (H2O2) and chloride to hypochlorous acid a strong oxidant with powerful antimicrobial activity and broad-spectrum reactivity with biomolecules MPO is considered an important marker for inammatory diseases autoimmune diseases and can-cer MPO is also experimentally and clinically important for distinguishing myeloid from lym-phoid leukemia and due to its role in the pathology of atherogenesis has been advocated as a prognostic marker of cardiovascular disease

e ferric or native MPO reacts with hydrogen H2O2 to form the active inter mediate MPO-I which oxidizes chloride (Clndash) to HOCl these reactions make up the chlorination cycle (Figure 18226) MPO also oxidizes a variety of substrates including phenols and anilines via the classic peroxidation cycle e relative concentrations of chloride and the reducing substrate determine whether MPO uses hydrogen peroxide for chlorination or peroxida-tion Assays based on measurement of chlorination activity are more specic for MPO than those based on peroxidase substrates such as tetramethylbenzidine (TMB)

Figure 18223 Absorption and uorescence emission spectra of resorun in pH 90 buer

Figure 18224 Detection of xanthine oxidase using the Amplexreg Red XanthineXanthine Oxidase Assay Kit (A22182) Each reaction contained 50 microM Amplexreg Red reagent 02 UmL horseradish peroxidase 01 mM hypo-xanthine and the indicated amount of xanthine oxidase in 1X reaction buer After 30 minutes uorescence was mea-sured in a uorescence microplate reader using excitation at 530 plusmn 125 nm and detection at 590 plusmn 175 nm A back-ground of 65 uorescence units was subtracted from each data point The inset shows the assayrsquos sensitivity and linear-ity at low hypoxanthine concentrations

Figure 18225 Detection of hypoxanthine using the Amplexreg Red XanthineXanthine Oxidase Assay Kit (A22182) Each reaction contained 50 microM Amplexreg Red reagent 02 UmL horseradish peroxidase 20 mUmL xan-thine oxidase and the indicated amount of hypoxanthine in 1X reaction buer Reactions were incubated at 37degC After 30 minutes uorescence was measured in a uorescence mi-croplate reader using excitation at 530 plusmn 125 nm and detec-tion at 590 plusmn 175 nm A background of 54 uorescence units was subtracted from each data point The inset shows the as-sayrsquos sensitivity and linearity at low enzyme concentrations

e Amplexreg Red XanthineXanthine Oxidase Assay Kit (A22182) contains

bull Amplexreg Red reagentbull Dimethylsulfoxide (DMSO)bull Horseradish peroxidase (HRP)bull H2O2bull Concentrated reaction buer

bull Xanthine oxidase from buttermilkbull Hypoxanthinebull Xanthinebull Detailed protocols








e EnzChekreg Myeloperoxidase (MPO) Activity Assay Kit (E33856) pro-vides assays for rapid and sensitive determination of both chlorination and peroxidation activities of MPO in solution and in cell lysates 159ndash161 (Figure 18226) For detection of chlorination the kit provides nonuorescent 3 -(p-aminophenyl) uorescein (APF) which is selectively cleaved by hypo-chlorite (ndashOCl) to yield uorescein Peroxidation is detected using nonuo-rescent Amplexreg UltraRed reagent which is oxidized by the H2O2-generated redox intermediates MPO-I and MPO-II to form a uorescent product e EnzChekreg Myeloperoxidase Activity Assay Kit can be used to continuously detect these activities at room temperature over a broad dynamic range (15 to 200 ngmL) (Figure 18227) e speed (30 minutes) sensitivity and mix-and-read convenience make this kit ideal for measuring MPO activities and for high-throughput screening for MPO-specic inhibitors

Each EnzChekreg Myeloperoxidase (MPO) Activity Assay Kit contains

bull 3 -(p-aminophenyl) uorescein (APF)bull Amplexreg UltraRed reagentbull Human myeloperoxidase (MPO) standardbull Chlorination inhibitorbull Peroxidation inhibitorbull Hydrogen peroxide (H2O2)bull Phosphate-buered saline (PBS)bull Dimethylsulfoxide (DMSO)bull Detailed protocols

Sucient reagents are provided to perform 200 assays for chlorination and 200 assays for peroxidation activity in a 96-well uorescence microplate format (100 microL per assay)

Zentrade Myeloperoxidase (MPO) ELISA Kite Zentrade Myeloperoxidase (MPO) ELISA Kit (Z33857) provides a com-

prehensive set of components for accurate and sensitive quantitation of hu-man MPO in a variety of biological samples including human serum is sandwich immunoassay utilizes the Amplexreg UltraRed reagent a uoro-genic substrate for horseradish peroxidase (HRP) that reacts with H2O2 in a 11 stoichiometric ratio to produce the brightly uorescent and strongly ab-sorbing Amplexreg UltraRed oxidation product (excitation emission maxima ~568581 nm) Because the Amplexreg UltraRed product has long-wavelength emission there is little interference from the blue or green autouorescence found in most biological samples With a high extinction coecient good quantum eciency and resistance to autooxidation the uorescence-based Amplexreg UltraRed reagent delivers better sensitivity and a broader assay range than colorimetric reagents

Each Zentrade Myeloperoxidase (MPO) ELISA Kit contains

bull Amplexreg UltraRed reagentbull Dimethylsulfoxide (DMSO)bull Concentrated phosphate-buered saline (PBS)bull Horseradish peroxidase (HRP) labeled goat antindashrabbit IgG antibodybull Amplexreg stop reagentbull Hydrogen peroxide (H2O2)bull MPO standardbull Bovine serum albumin (BSA)bull Tweenreg 20bull Mouse anti-MPO antibody (capture antibody)bull Rabbit anti-MPO antibody (detection antibody)bull Zentrade microplates for oriented capture anitbody coatingbull Detailed protocols

Figure 18226 Schematic diagram for detection of chlorination and peroxidation activity of MPO using the EnzChekreg Myeloperoxidase (MPO) Activity Assay Kit (E33856) AH2 rep-resents the nonuorescent Amplexreg UltraRed substrate and A represents its uorescent oxidation product

uorescein3prime-(p-aminophenyl) uorescein (APF)

HOCl Clndash

H2O2

AH2

AH2A

AH2

AH

AH

AH

AH+ +

MPO-II

MPO-IMPOChlorination activity

Peroxidation activity

Figure 18227 Typical standard curves for detection of MPO using the APF-based chlorination assay (A) and Amplexreg UltraRedndashbased peroxidation assay (B) provided in the EnzChekreg Myeloperoxidase (MPO) Activity Assay Kit (E33856) Reactions were incubated at room temperature for 30 minutes Values on the x-axes are concentra-tions of MPO in the standards prior to adding the detection reagent Fluorescence was measured with a uorescence microplate reader using uorescence excitation and emission at 485 and 530 nm respectively for the APF assay or excitation and emission at 530 and 590 nm respectively for the Amplexreg UltraRed assay The back-ground uorescence measured for each zero-MPO control reaction was subtracted from each uorescence measurement before plotting

0 1601208040 200

1400

0

2800

4200

5600

7000

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 963 120

100

200

00

40 80 120 160 200

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

4000

3200

2400

1600

800

0 129630

100

200

A

B







Sucient reagents are provided for 200 assays in a microplate format using a 100 microL per well reaction volume e Zentrade Myeloperoxidase (MPO) ELISA Kit can be used to detect from 02 to 100 ngmL MPO at room temperature (Figure 18228)

Assaying Oxidative Activity in Live Cellse 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 reperfu-sion injury and neuorodegenerative disorders40162163

Assaying oxidative activity in live cells with uorogenic chemiluminescent or chromogenic probes is complicated by the frequent presence of multiple reactive oxygen species in the same cell Scavengers and enzymes such as superoxide dismutase and catalase are useful knockdown reagents for triaging the optical response of ROS probes (Table 182) Quantitative analysis can be further hindered due to 1) the high intracellular concentration of glutathione which can form thiyl or sulnyl radicals or otherwise trap or reduce oxygen species 164 2) the variable concentra-tion of metals which can either catalyze or inhibit radical reactions and 3) the presence of other free radicalndashquenching agents such as spermine165

Fluorescein rhodamine and various other dyes can be chemically reduced to colorless nonuorescent leuco dyes ese dihydro derivatives are readily oxidized back to the parent dye by reactive oxygen species and thus can serve as uorogenic probes for detecting oxidative activity in cells and tissues166ndash168 Oxidation also occurs spontaneously albeit slowly in air and via photosensitization when illuminated for uorescence excitation169170 Careful storage and handling as well as minimizing the duration and intensity of light exposure are particularly recommended when using these dyes In general dihydrouorescein and dihydrorhodamine do not discriminate between the various reactive oxygen species It has been reported that dichlo-rodihydrouorescein (H2DCF) and dihydrorhodamine 123 react with intracellular hydrogen peroxide in a reaction mediated by peroxidase cytochrome c or Fe2+23171 and these leuco dyes also serve as uorogenic substrates for peroxidase enzymes (Section 105)

Dichlorodihydrouorescein Diacetatee cell-permeant 2 7 -dichlorodihydrouorescein diacetate (H2DCFDA D399 Figure

18229) also known as dichlorouorescin diacetate is commonly used to detect the generation of reactive oxygen intermediates in neutrophils and macrophages172ndash176 Upon cleavage of the ac-etate groups by intracellular esterases and subsequent oxidation the nonuorescent H2DCFDA is converted to the highly uorescent 2 7 -dichlorouorescein (DCF)

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 sub-strates that yield peroxy products and peroxyl radicals170 In a cell-free system H2DCF has been shown to be oxidized to DCF by peroxynitrite anion (ONOOndash) by horseradish peroxidase (in the absence of H2O2) and by Fe2+ (in the absence of H2O2)177 Furthermore the oxidation of H2DCF by Fe2+ in the presence of H2O2 was reduced by the HObull radical scavenger formate and the iron chelator deferoxamine177 In addition DCF itself can act as a photosensitizer for H2DCFDA oxidation both priming and accelerating the formation of DCF170 Because the oxidation of DCF and H2DCFDA appears to also generate free radicals their use for measuring free radical production must be carefully controlled178

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 P21493 Section 81) with H2DCFDA to simultaneously monitor oxidant production and cell injury179 H2DCFDA has been used to visualize oxidative changes in carbon tetrachloridendashperfused rat liver 180 and in venular endothelium during neutrophil activation181 as well as to examine the eect of ischemia and reperfusion in lung and heart tissue182183 Using H2DCFDA researchers characterized hypoxia-dependent peroxide production in Saccharomyces cerevisiae as a possible model for ischemic tissue destruction184 In neutrophils H2DCFDA has proven

Figure 18228 Typical standard curve for detection of MPO using the Zentrade Myeloperoxidase (MPO) ELISA Kit (Z33857) The sandwich ELISA was carried out as described in the protocol using a mouse anti-MPO primary capture an-tibody MPO standards ranging from 02 ngmL to 100 ngmL and a rabbit anti-MPO detection antibody

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0

Figure 18229 2rsquo7rsquo-dichlorodihydrouorescein diacetate (2rsquo7rsquo-dichlorouorescin diacetate H2DCFDA D399)








useful for ow cytometric analysis of nitric oxide forming a product that has spectral properties identical to those produced when it reacts with hydrogen peroxide185 In this study H2DCFDArsquos reaction with nitric oxide was blocked by adding the nitric oxide synthase inhibitor NG-methyl-L-arginine (L-NMMA) to the cell suspension185 2 7 -Dichlorouoresceinmdashthe oxidation prod-uct of H2DCFmdashcan reportedly be further oxidized to a phenoxyl radical in a horseradish peroxi-dasendashcatalyzed reaction and this reaction may complicate the interpretation of results obtained with this probe in cells undergoing oxidative stress186 Although other more specialized ROS probes have beenmdashand continue to bemdashdeveloped H2DCFDA and its chloromethyl derivative CM-H2DCFDA remain the most versatile indicators of cellular oxidative stress187

Improved Versions of H2DCFDAIntracellular oxidation of H2DCF tends to be accompanied by leakage of the product 2 7 -di-

chlorouorescein188 which may make quantitation or detection of slow oxidation dicult To en-hance retention of the uorescent product we oer the carboxylated H2DCFDA analog 189 (carboxy-H2DCFDA C400) which has two negative charges at physiological pH and its di(acetoxymethyl ester) analog 6-carboxy-2rsquo7rsquo-dichlorodihydrouorescein diacetate di(acetoxymethyl ester) 190 (C2938 Figure 18230 Figure 18231) Upon cleavage of the acetate and ester groups by intracel-lular esterases and oxidation both analogs form carboxydichlorouorescein (C368 Section 143) with additional negative charges that impede its leakage out of the cell

e uorinated analog 5-(and 6-)carboxy-2 7 -diuorodihydrouorescein diacetate (car-boxy-H2DFFDA C13293) is also useful for visualizing oxidative bursts and inammatory and infectious processes191 As the oxidation potential of deacetylated carboxy-H2DFFDA is more positive than that of the corresponding chloro compound carboxy-H2DCFDA its oxidant sen-sitivity prole is presumably shied however it is not known if this dierence is large enough to have practical utility e diacetate derivatives of the dichloro- and diuorodihydrouoresceins are quite stable When used for intracellular applications the acetates are cleaved by endogenous esterases releasing the corresponding dichloro- or diuorodihydrouorescein derivative If however these nonuorescent diacetate derivatives are used for in vitro assays they must rst be hydrolyzed with mild base to form the colorless probe150

In addition we have developed 5-(and 6-)chloromethyl-2 7 -dichlorodihydrouorescein diacetate acetyl ester (CM-H2DCFDA C6827 Figure 18232 Figure 18233) which is a chloro-methyl derivative of H2DCFDA that exhibits much better retention in live cells192193 As with our other chloromethyl derivatives (see the description of our CellTrackertrade probes in Section 142) CM-H2DCFDA passively diuses 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 uorescent adduct that is trapped inside the cell thus facili-tating long-term studies192193 Among its many applications CM-H2DCFDA has been used to

bull Analyze FOXO3 transcriptional control of oxidative stress 194195

bull Assess ROS-mediated cytotoxicity and apoptosis 196ndash199

bull Detect hydroxyl radicals associated with estrogen-induced DNA damage 200

bull Monitor time courses of ROS generation in neurons and brain slices 192201

bull Analyze ROS production in chromosomally unstable humanndashhamster hybrid cells using ow cytometry 202

Figure 18230 6-carboxy-2rsquo7rsquo-dichlorodihydrouorescein diacetate di(acetoxymethyl ester) C2938

O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O

Figure 18231 Bovine pulmonary artery endothelial (BPAEC) cells were initially stained with the reactive oxygen species (ROS) indicator 6-carboxy-2rsquo7rsquo-dichlorodihydro-uorescein diacetate di(acetoxymethyl ester) (C2938) After a 30-minute incubation the cells were washed and then incubated simultaneously with FMreg 5-95 (T23360) and Hoechst 33342 (H1399 H3570 H21492) in phosphate-bu-ered saline (PBS) for an additional 5 minutes before washing and mounting in PBS The red-uorescent FMreg 5-95 appears to stain both the plasma membrane and early endosomes the green-uorescent oxidized carboxydichlorouores-cein localizes to the cytoplasm and the blue-uorescent Hoechst 33342 dye stains the nucleus

Figure 18232 5-(and-6)-chloromethyl-2rsquo7rsquo-dichlorodihydro-uorescein diacetate acetyl ester (CM-H2DCFDA C6827)

CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO

Figure 18233 An oxidative burst was detected by ow cytometry of cells labeled with 5-(and 6-)chloromethyl-2rsquo7rsquo-dichlorodihydrou-orescein diacetate acetyl ester (CM-H2DCFDA C6827) Jurkat cells were incubated with 100 nM CM-H2DCFDA The cells were washed and resuspended in either phosphate-buered saline (PBS red) or PBS with 003 H2O2 (blue) The samples were analyzed on a ow cytometer equipped with a 488 nm argon-ion laser and a 525 plusmn 10 nm bandpass emission lter

100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0







Image-iTreg LIVE Green Reactive Oxygen Species Detection Kite Image-iTreg LIVE Green Reactive Oxygen Species Detection Kit (I36007) provides the

key reagents for detecting reactive oxygen species (ROS) in live cells (Figure 18234) including

bull Carboxy-H2DCFDA (5-(and 6-)carboxy-2 7 -dichlorodihydrouorescein diacetate)bull Hoechst 33342bull tert-butyl hydroperoxide (TBHP)bull Dimethylsulfoxide (DMSO)bull Detailed protocols for uorescence microscopy assays

is assay is based on carboxy-H2DCFDA (5-(and 6-)carboxy-2 7 -dichlorodihydrouorescein diacetate) a reliable uorogenic marker for reactive oxygen species in live cells203204 In addition to carboxy-H2DCFDA this kit provides the common inducer of ROS production tert-butyl hydro-peroxide (TBHP) as a positive control 205ndash208 and the blue-uorescent cell-permeant nucleic acid stain Hoechst 33342 Oxidatively stressed and nonstressed cells can be eectively distinguished by uorescence microscopy using this combination of dyes and the protocol provided209ndash211

Aminophenyl Fluorescein and Hydroxyphenyl FluoresceinDeveloped by Nagano 3 -(p-aminophenyl) uorescein (APF A36003) and 3 -(p-hydroxy-

phenyl) uorescein (HPF H36004) provide greater selectivity and stability than dichlorodihy-drouorescein diacetate (H2DCFDA D399) for ROS detection212 H2DCFDA is probably the most commonly used reagent for detecting intracellular reactive oxygen species despite its lack of specicity and tendency to spontaneously photooxidize e nonuorescent H2DCFDA becomes uorescent in the presence of a wide variety of reactive oxygen species including but not lim-ited to peroxyl (ROObull) and hydroxyl (HObull) radicals and the peroxynitrite anion (ONOOndash) In contrast APF and HPF show much more limited reactivity and greater resistance to light-in-duced oxidation (Table 184) Both of these uorescein derivatives are essentially nonuores-cent until they react with the hydroxyl radical60 peroxynitrite anion or singlet oxygen 16 (Figure 18235) APF will also react with the hypochlorite anion (ndashOCl) making it possible to use APF

Figure 18234 Detection of oxidative stress in live cells using the Image-iTreg LIVE Green Reactive Oxygen Species (ROS) Detection Kit (I36007) Live bovine pulmonary artery endothelial cells were treated with tert-butyl hydroperoxide to induce oxidative stress (bottom) or were left untreated (top) Cells were then labeled with carboxy-H2DCFDA which uoresces when oxidized by ROS and nuclei were stained with blue-uorescent Hoechst 33342 The stressed cells exhibited green uorescence signaling an increase in ROS whereas the untreated cells showed minimal uorescence

Table 184 Fluorescence response of APF HPF and H2DCFDA to various reactive oxygen species (ROS)

Reactive Oxygen Species (ROS) ROS Generation Method APF HPF H2DCFDA

Hydrogen peroxide (H2O2) 100 microM H2O2 lt1 2 190

Hydroxyl radical (HObull) 100 microM ferrous perchlorate (II) and 1 mM of H2O2 1200 730 7400

Hypochlorite anion (ndashOCl) 3 microM (nal) ndashOCl 3600 6 86

Nitric oxide (NO) 100 microM 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-methyl-1-triazene (NOC-7) lt1 6 150

Peroxyl radical (ROObull) 100 microM 22rsquo-azobis(2-amidinopropane) dihydrochloride (AAPH) 2 17 710

Peroxynitrite anion (ONOOndash) 3 microM (nal) ONOO 560 120 6600

Singlet oxygen (1O2) 100 microM 3-(14-dihydro-14-epidioxy-1-naphthyl)propionic acid 9 5 26

Superoxide anion (bullO2ndash) 100 microM KO2 6 8 67

Autooxidation 25 hours exposure to uorescent light source lt1 lt1 2000

10 microm of APF HPF or DCF (2rsquo7rsquo-dichlorouorescein) were added to sodium phosphate buer (01 M pH 74) ROS were generated as indicated and uorescence was measured using excitationemission wavelengths of 490515 nm (for APF and HPF) or 500520 nm (for DCF) DCF was obtained by hydrolysis of H2DCFDA with base as described in J Biol Chem (2003) 2783170 dihydrouorescein diacetates are colorless and nonuorescent until both of the acetate groups are hydrolyzed and the products are subsequently oxidized to uorescein derivatives

Figure 18235 Detection of reactive oxygen species (ROS) with 3rsquo-(p-hydroxyphenyl) uorescein (HPF H36004) and 3rsquo-(p-aminophenyl) uorescein (APF A36003)

COO

OO O

XH

COO

OO O

OX

ROS

Nonuorescent Fluorescent

X = O 3ʹ-(p-hydroxyphenyl) uorescein (HPF)3ʹ-(p-aminophenyl) uorescein (APF)X = NH








and HPF together to selectively detect the hypochlorite anion (Section 212) In the presence of these specic reactive oxygen species both APF and HPF yield a bright green-uorescent product (excitationemission maxima ~490515 nm) and are compatible with all uorescence instrumentation capable of visualizing uorescein 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 indicators212

Dihydrocalcein AMWe have combined the superior retention of calcein (the intracellular product of calcein

AM hydrolysis in viable cells) and the oxidation sensitivity of the dihydrouoresceins to yield the probe dihydrocalcein AM (D23805 Figure 18236) provided specially packaged as a set of 20 vials each containing 50 microg e oxidant sensitivity prole of dihydrocalcein AM has been characterized relative to that of H2DCFDA 213 and of alkaline elution assays of oxidative DNA modication214

OxyBURSTreg Green ReagentsFc OxyBURSTreg Green assay reagent (F2902) was developed in collaboration with Elizabeth

Simons of Boston University to monitor the oxidative burst in phagocytic cells using uorescence instrumentation e Fc OxyBURSTreg Green assay reagent comprises bovine serum albumin (BSA) that has been covalently linked to dichlorodihydrouorescein (H2DCF) and then com-plexed with puried rabbit polyclonal anti-BSA antibodies When these immune complexes bind to Fc receptors the nonuorescent H2DCF molecules are internalized within the phagovacuole and subsequently oxidized to green-uorescent dichlorouorescein (DCF) see Section 161 for a more complete description

OxyBURSTreg Green H2HFF BSA (O13291) is a sensitive uorogenic reagent for de-tecting extracellular release of oxidative products in a spectrouorometer or a uores-cence microscope is reagent comprises BSA that has been covalently linked to dihydro-2 45677 -hexauorouorescein (H2HFF) a reduced dye with improved stability Unlike Fc OxyBURSTreg Green assay reagent OxyBURSTreg Green H2HFF BSA is not complexed with IgG OxyBURSTreg Green H2HFF BSA provides up to 1000-fold greater sensitivity than conven-tional methods based on spectrophotometric detection of superoxide dismutasendashinhibitable re-duction of cytochrome c215216

Amine-Reactive OxyBURSTreg Green ReagentAs an alternative to Fc OxyBURSTreg Green assay reagent and OxyBURSTreg Green H2HFF BSA

we oer the amine-reactive OxyBURSTreg Green H2DCFDA succinimidyl ester (2 7 -dichloro-dihydrouorescein diacetate SE D2935 Figure 18237) which can be used to prepare oxida-tion-sensitive conjugates of a wide variety of biomolecules and particles including antibod-ies antigens peptides proteins dextrans bacteria yeast and polystyrene microspheres217218 Following conjugation to amines the two acetates of OxyBURSTreg Green H2DCFDA can be removed by treatment with hydroxylamine at neutral pH to yield the dihydrouorescein conju-gate OxyBURSTreg Green H2DCFDA conjugates are nonuorescent until they are oxidized to the corresponding uorescein derivatives

Dihydrorhodamine 123Dihydrorhodamine 123 (D632 D23806 Figure 18238 Figure 18239) is the uncharged

and nonuorescent reduction product of the mitochondrion-selective dye rhodamine 123 (R302 R22420 Section 122) is leuco dye passively diuses across most cell membranes where it is oxidized to cationic rhodamine 123 (Figure 18240) which localizes in the mitochondria Like H2DCF dihydrorhodamine 123 does not directly detect superoxide219 but rather reacts with hydrogen peroxide in the presence of peroxidase cytochrome c or Fe2+23 However dihydrorho-damine 123 also reacts with peroxynitrite the anion formed when nitric oxide reacts with super-oxide171 Peroxynitrite which may play a role in many pathological conditions has been shown to react with sulydryl groups DNA and membrane phospholipids as well as with tyrosine and other phenolic compounds220ndash223

Dihydrorhodamine 123 has been used to investigate reactive oxygen intermediates pro-duced by human and murine phagocytes45 activated rat mast cells 224 and vascular endothelial

Figure 18237 2rsquo7rsquo-dichlorodihydrouorescein diacetate succinimidyl ester (OxyBURSTreg Green H2DCFDA SE D2935)

O OCCH3

CH

C C

CH3COO O

O

O

OO

Figure 18239 Live bovine pulmonary artery endothe-lial cells (BPAEC) were rst stained with LysoTrackerreg Red DND-99 (L7528) Then a solution of dihydrorhodamine 123 (D632 D23806) and Hoechst 33258 (H1398 H3569 H21491) was added and allowed to incubate with the cells for an additional 10 minutes before the cells were subsequently washed and visualized The green-uorescent oxidation product (rhodamine 123 R302) localized primarily to the mitochondria The red-uorescent LysoTrackerreg Red DND-99 stain accumulated in the lysosomes and the blue-u-orescent Hoechst 33258 dye stained the nuclei The image was acquired with lters appropriate for DAPI uorescein and the Texas Redreg dye The image was deconvolved us-ing Huygens software (Scientic Volume Imaging wwwsvinl) 3D reconstruction was performed using Imaris software (Bitplane AG wwwbitplanecom)

Figure 18238 Dihydrorhodamine 123 (D632)

Figure 18236 Dihydrocalcein AM (D23805)

CH3CO O OCCH3

COCHOCCH3

CH CH

CHCOCHOCCH3CH3COCHOCCHO O

O OO O

OO

H







tissues225226 It has also been employed to study the role of the CD14 cell-surface marker in H2O2 production by human monocytes227

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 RhodamineIntracellular oxidation of dihydrorhodamine 6G (D633) yields rhodamine 6G (R634) which

localizes in the mitochondria of live cells (Section 122) As compared with rhodamine 123 this cationic oxidation product has longer-wavelength spectra making it especially useful in multi-color applications and in autouorescent cells and tissues Dihydrorhodamine 6G has been used for uorescence microplate assays of granulocyte activation 228 and for analysis of ROS levels in human umbilical vein endothelial (HUVEC) cells by ow cytometry194

Reduced MitoTrackerreg ProbesTwo of our MitoTrackerreg probesmdashMitoTrackerreg Orange CM-H2TMRos (M7511 Figure

18241) and MitoTrackerreg Red CM-H2XRos (M7513 Figure 18242)mdashare chemically reactive reduced rosamines Unlike MitoTrackerreg Orange CMTMRos and MitoTrackerreg Red CMXRos (M7510 M7512 Section 122) the reduced versions of these probes do not uoresce until they enter an actively respiring cell where they are oxidized by reactive oxygen species to the uo-rescent mitochondrion-selective probe and then sequestered in the mitochondria Although CM-H2TMRos and CM-H2XRos are widely used as indicators of mitochondrial reactive oxygen species229ndash231 their uorescence cannot be unambiguously associated with the site of oxidant generation as the cationic charge that drives their electrophoretic sequestration in active mito-chondria is only present aer the probe has been oxidized is same caveat also applies to di-hydrorhodamine 123 and dihydrorhodamine 6G Probes such as MitoSOXtrade Red mitochondrial superoxide indicator resolve this ambiguity by having their oxidant response and mitochondrial localization functions associated with dierent structural elements (Figure 1828)

RedoxSensortrade Red CC-1 StainRedoxSensortrade Red CC-1 stain (23456-pentauorotetramethyldihydrorosamine R14060

Figure 18243) passively enters live cells and is subsequently oxidized in the cytosol to a red-uorescent product (excitationemission maxima ~540600 nm) which then accumulates in the mitochondria Alternatively this nonuorescent probe may be transported to the lysosomes where it is oxidized e dierential distribution of the oxidized product between mitochondria and lysosomes appears to depend on the redox potential of the cytosol232ndash234 In proliferating cells mitochondrial staining predominates whereas in contact-inhibited cells the staining is primarily lysosomal (Figure 18244)

Glutathiolation Detection with BioGEEBiotinylated glutathione ethyl ester (BioGEE G36000 Figure 18245) is a cell-permeant

biotinylated glutathione analog for the detection of glutathiolation Under conditions of oxi-dative stress cells may transiently incorporate glutathione into proteins Stressed cells incu-bated with BioGEE will also incorporate this biotinylated glutathione derivative into proteins facilitating the identication of oxidation-sensitive proteins235236 Once these cells are xed and

Figure 18240 Rhodamine 123 (R302)

H O H

CO

OCH3

C

Figure 18241 MitoTrackerreg Orange CM-H2TMRos (M7511)

(CH3)2N O N(CH3)2

CH2Cl

H

Figure 18242 MitoTrackerreg Red CM-H2XRos (M7513)

O

CHC

H

Figure 18243 RedoxSensortrade Red CC-1 (R14060)

CH3 O CH3

H

Figure 18244 Cellular proliferation state determines the distribution of the oxidized product of RedoxSensortrade Red CC-1 (R14060) Normal rat kidney (NRK) cells in dierent growth states were stained with RedoxSensortrade Red CC-1 In proliferating cells (left panel) the oxidized dye accumulates in mitochondria In quiescent cells (right panel) the oxi-dized product localizes in the lysosomesFigure 18245 Glutathione ethyl ester biotin amide (BioGEE G36000)

CHCHCHCHCH

O

H

H

OH

H

H

C HCH C HCH C OCHCH3

O O O

CHHCOH

O








permeabilized glutathiolation levels can be detected with a uorescent streptavidin conjugate (Section 76 Table 79) using either ow cytometry or uorescence microscopy Proteins glutathi-olated with BioGEE can be captured using streptavidin agarose (S951 Section 76) and analyzed by mass spectrometry or by western blotting methods198237

Tetrazolium Salts Chromogenic Redox IndicatorsTetrazolium saltsmdashespecially MTT (M6494 Figure 18246)mdashare 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 aer solubilization in organic solvent for quantitation by standard spectro-photometric techniques e extremely water-soluble formazan product of XTT (X6493) does not require solubilization prior to quantitation

Selected applications of the tetrazolium salts are listed in Table 183 Our Vybrantreg MTT Cell Proliferation Assay Kit (V13154 Section 154) provides a means of counting metabolically active cells this Vybrantreg MTT assay can detect from 2000 to 250000 cells depending on the cell type and conditions See also Section 152 for additional cell applications of tetrazolium salts

Figure 18246 MTT (3-(45-dimethylthiazol-2-yl)-25-diphenyl-tetrazolium bromide) M6494

CH3

CH3

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202 Cancer Res (2003) 633107 203 Am J Physiol Heart Circ Physiol (2000) 279H2424 204 J Natl Cancer Inst (1999) 911138 205 Lipids (2001) 3657 206 Cancer Res (2001) 611392 207 Histochem Cell Biol (2003) 120319 208 Am J Physiol (1997) 272C1286 209 Nature (2007) 447686 210 J Neurochem (2006) 981474 211 J Biol Chem (2006) 2816760 212 J Biol Chem (2003) 2783170 213 Free Radic Res (2004) 381257 214 Toxicol In Vitro (2007) 211552 215 J Biol Chem (1980) 2551874 216 J Clin Invest (1978) 611081 217 J Cell Physiol (1993) 156428 218 J Immunol Methods (1990) 130223 219 Eur J Biochem (1993) 217973 220 Free Radic Res (2000) 33771 221 Methods Mol Biol (1998) 100215 222 Mol Med (2000) 6779 223 Methods Enzymol (1994) 233229 224 APMIS (1994) 102474 225 Atherosclerosis (2003) 16919 226 Circ Res (2004) 94239 227 J Immunol Methods (2006) 31627 228 Nat Med (2009) 15300 229 Nat Clin Pract Cardiovasc Med (2008) 5811 230 Biochemistry (2006) 457237 231 Diabetes (2006) 55120 232 Am J Pathol (2009) 174101 233 Am J Physiol Renal Physiol (2007) 292F523 234 Free Radic Biol Med (2000) 281266 235 Amino Acids (2007) 3351 236 Biochemistry (2000) 3911121 237 J Biol Chem (2009) 28422213

REFERENCESmdashcontinued

DATA TABLE 182 GENERATING AND DETECTING REACTIVE OXYGEN SPECIESCat No MW Storage Soluble Abs EC Em Solvent NotesA7923 37648 FDL DMSO 358 11000 424 MeOH 1A12222 25725 FFDA DMSO 280 6000 none pH 8 2A22177 25725 FFDA DMSO 280 6000 none pH 8A36003 42342 ROL DMF 454 24000 515 pH 9 3 4A36006 ~300 FFDA DMSO 293 11000 none pH 8 5B3932 44832 FL DMSO CHCl3 665 161000 676 MeOHC400 53130 FD DMSO EtOH 290 5600 none MeCN 6C2938 67543 FDAA DMSO 291 5700 none MeOH 6C2944 42347 FFDLLAA MeOH 429 7500 see Notes pH 7 7 8 9C6827 57780 FDAA DMSO 287 9100 none MeOH 6C7924 48762 FDL DMSO H2O 385 5800 485 pH 7 1C13293 49839 FD DMSO EtOH 290 5500 none MeCN 10D399 48729 FD DMSO EtOH 258 11000 none MeOH 6D632 34638 FDLAA DMF DMSO 289 7100 none MeOH 11 12D633 44457 FDLAA DMF DMSO 296 11000 none MeOH 11 12D1168 31542 FFLAA DMF DMSO 355 14000 see Notes MeCN 11 13D2935 58437 FDAA DMF 258 11000 none MeOH 6D3861 50443 FL DMSO 582 140000 591 MeOH 14D7894 38643 FDLL MeCN 358 29000 none MeOH 15D11347 31542 FFLAA DMF DMSO 355 14000 see Notes MeCN 11 13D23107 31542 FFDLAA DMSO 355 14000 see Notes MeCN 13 16D23805 106895 FD DMSO 285 5800 none MeCN 17D23806 34638 FDLAA DMSO 289 7100 none MeOH 12 16F2902 see Notes RRLAA H2O lt300 none 3 18 19G36000 56167 FD DMSO lt300 noneH7476 50445 FDL DMSO DMF 591 37000 594 EtOHH36004 42441 ROL DMF 454 28000 515 pH 9 3 4L6868 51050 L H2O 455 7400 505 H2O 20 21L8455 17716 DL DMF 355 7500 411 MeOH 21M689 48598 FDDL DMF DMSO 629 75000 none MeCN 22M6494 41432 DL H2O DMSO 375 8300 none MeOH 23 24M7511 39293 FDLAA DMSO 235 57000 none MeOH 11 12M7513 49708 FDLAA DMSO 245 45000 none MeOH 11 12M7913 25832 FL DMF DMSO 352 30000 401 MeOH 25M23800 29174 FFDLLAA DMSO 430 8400 546 MeOH 26M24571 56967 DL DMSO EtOH 555 143000 578 MeOHM36008 75971 FFLAA DMSO 356 10000 410 MeCN 11 27N6495 81765 DL H2O DMSO 256 64000 none MeOH 23O13291 ~66000 FDLAA H2O lt300 none 28P800 ~240000 RRL see Notes 546 2410000 575 pH 7 29P801 ~240000 RRL see Notes 565 1960000 578 pH 7 29

continued on next page








P6879 36316 FDL DMSO 270 28000 none CHCl3 30P36005 27642 FFLLAA EtOH 304 77000 416 MeOH 3 31R14000 105775 FD DMSO 313 9700 none MeOH 32R14060 43441 FDLAA DMSO 239 52000 none MeOH 11 33S36002 ~600 FDL DMSO 508 105000 528 pH 7 34 35X6493 67453 FD H2O DMSO 286 15000 none MeOH 36For denitions of the contents of this data table see ldquoUsing The Molecular Probesreg Handbookrdquo in the introductory pagesNotes

1 Fluorescence of A7923 and C7924 is weak Reaction of the nitroxide moiety with superoxide or hydroxyl radicals results in increased uorescence without a spectral shift (Anal Biochem (1993) 21285)

2 Peroxidase-catalyzed reaction of the Amplexreg Red reagent (A12222 A22177) with H2O2 produces uorescent resorun (R363) Resorun is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol (Bioorg Chem (1998) 2663)

3 This product is supplied as a ready-made solution in the solvent indicated under Soluble4 Fluorescence of A36003 and H36004 is extremely weak Highly uorescent uorescein F1300 is generated upon oxidation (J Biol Chem (2003) 2783170)5 Peroxidase-catalyzed reaction of the Amplexreg UltraRed reagent (A36006) with H2O2 yields a uorescent product with Abs = 568 nm (EC = 57000 cmndash1Mndash1) Em = 581 nm in pH 75 buer6 Dihydrouorescein diacetates are colorless and nonuorescent until both of the acetate groups are hydrolyzed and the products are subsequently oxidized to uorescein derivatives The materi-

als contain less than 01 of oxidized derivative when initially prepared The oxidation products of C400 C2938 C6827 D399 and D2935 are 2acute7acute-dichlorouorescein derivatives with spectra similar to C368 (Section 143)

7 C2944 emits chemiluminescence (Em = 466 nm) on oxidation by superoxide (Anal Biochem (1992) 206273)8 Do NOT dissolve in DMSO9 Aqueous solutions of coelenterazine (gt1 mM) can be prepared in pH 7 buer containing 50 mM 2-hydroxypropyl-β-cyclodextrin (Biosci Biotechnol Biochem (1997) 611219)10 Diuorodihydrouorescein diacetates are colorless and nonuorescent Acetate hydrolysis and subsequent oxidation generate a uorescent 2acute7acute-diuorouorescein derivative with spectra

similar to O6146 (Section 143)11 This compound is susceptible to oxidation especially in solution Store solutions under argon or nitrogen Oxidation may be induced by illumination12 These compounds are essentially colorless and nonuorescent until oxidized Oxidation products (in parentheses) are as follows D632 and D23806 (R302) D633 (R634) M7511 (M7510) M7513

(M7512)13 Dihydroethidium has blue uorescence (Em ~420 nm) until oxidized to ethidium (Em ~605 nm) The reduced dye does not bind to nucleic acids (FEBS Lett (1972) 26169)14 Oxidation of the polyunsaturated butadienyl portion of the BODIPYreg 581591 dye results in a shift of the uorescence emission peak from ~590 nm to ~510 nm (Methods Enzymol (2000)

319603 FEBS Lett (1999) 453278)15 Oxidation of D7894 occurs rapidly in solution when illuminated The oxidation product is strongly uorescent Em = 379 nm16 This product is supplied as a ready-made solution in DMSO with sodium borohydride added to inhibit oxidation17 D23805 is colorless and nonuorescent until the AM ester groups are hydrolyzed and the resulting leuco dye is subsequently oxidized The nal product is calcein (C481)18 F2902 is essentially colorless and nonuorescent until oxidized A small amount (~5) of oxidized material is normal and acceptable for the product as supplied The oxidation product is

uorescent (Abs = 495 nm Em = 524 nm) (J Immunol Methods (1990) 130223)19 This product consists of a dyendashbovine serum albumin conjugate (MW ~66000) complexed with IgG in a ratio of approximately 14 molmol (BSAIgG)20 L6868 has much stronger absorption at shorter wavelengths (Abs = 368 nm (EC = 36000 cmndash1Mndash1))21 This compound emits chemiluminescence upon oxidation in basic aqueous solutions Emission peaks are at 425 nm (L8455) and 470 nm (L6868)22 Isothiocyanates are unstable in water and should not be stored in aqueous solution23 Enzymatic reduction products are water-insoluble formazans with Abs = 505 nm (M6494) and 605 nm (N6495) after solubilization in DMSO or DMF See literature sources for further informa-

tion (Histochemistry (1982) 76381 Prog Histochem Cytochem (1976) 91)24 M6494 also has Abs = 242 nm (EC = 21000 cmndash1Mndash1) in MeOH25 Generates chemiluminescence (Em = 465 nm in 01 M SDS) upon reaction with 1O2 (J Am Chem Soc (1986) 1084498)26 Generates chemiluminescence (Em = 455 nm) upon reaction with superoxide27 Spectroscopic properties of the product generated by reaction of M36008 with superoxide are described in Nat Protoc (2008) 3828 Oxidation of O13291 generates a uorescent protein conjugate (Abs ~508 nm Em ~528 nm)29 Phycobiliproteins are packaged as suspensions in 60 ammonium sulfate pH 70 Store refrigerated at 4degC but DO NOT FREEZE30 Iodoacetamides in solution undergo rapid photodecomposition to unreactive products Minimize exposure to light prior to reaction31 Cis-parinaric acid is readily oxidized to nonuorescent products Use under N2 or Ar except when oxidation is intended Stock solutions should be prepared in deoxygenated solvents

Cis-parinaric acid is appreciably uorescent in lipid environments and organic solvents but is nonuorescent in water32 Acetate hydrolysis of R14000 yields rose bengal (Abs = 556 nm (EC = 104000 cmndash1Mndash1) Em = 572 nm in MeOH) (Photochem Photobiol (1997) 66374)33 R14060 is colorless and nonuorescent until oxidized The spectral characteristics of the oxidation product (23456-pentauorotetramethylrosamine) are similar to those of T639 (Section 122)34 MW The preceding ~ symbol indicates an approximate value not including counterions35 The uorescence of S36002 is relatively weak Reaction of the dye with singlet oxygen (1O2) results in uorescence enhancement with essentially no change in absorption or emission wave-

lengths36 Enzymatic reduction product is a water-soluble formazan Abs = 475 nm

DATA TABLE 182 GENERATING AND DETECTING REACTIVE OXYGEN SPECIESmdashcontinuedCat No MW Storage Soluble Abs EC Em Solvent Notes







PRODUCT LIST 182 GENERATING AND DETECTING REACTIVE OXYGEN SPECIESCat No Product QuantityA7923 4-((9-acridinecarbonyl)amino)-2266-tetramethylpiperidin-1-oxyl free radical (TEMPO-9-AC) 5 mgA36003 3acute-(p-aminophenyl) uorescein (APF) 5 mM solution in DMF 470 microLA22188 Amplexreg Red Hydrogen PeroxidePeroxidase Assay Kit 500 assays 1 kitA12222 Amplexreg Red reagent 5 mgA22177 Amplexreg Red reagent packaged for high-throughput screening 10 x 10 mgA22182 Amplexreg Red XanthineXanthine Oxidase Assay Kit 400 assays 1 kitA36006 Amplexreg UltraRed reagent 5 x 1 mgB3932 (EE)-35-bis-(4-phenyl-13-butadienyl)-44-diuoro-4-bora-3a4a-diaza-s-indacene (BODIPYreg 665676) 5 mgC400 5-(and-6)-carboxy-2acute7acute-dichlorodihydrouorescein diacetate (carboxy-H2DCFDA) mixed isomers 25 mgC2938 6-carboxy-2acute7acute-dichlorodihydrouorescein diacetate di(acetoxymethyl ester) 5 mgC13293 5-(and-6)-carboxy-2acute7acute-diuorodihydrouorescein diacetate (carboxy-H2DFFDA) mixed isomers 5 mgC7924 5-(2-carboxyphenyl)-5-hydroxy-1-((2255-tetramethyl-1-oxypyrrolidin-3-yl)methyl)-3-phenyl-2-pyrrolin-4-one potassium salt (proxyl uorescamine) 5 mgC6827 5-(and-6)-chloromethyl-2acute7acute-dichlorodihydrouorescein diacetate acetyl ester (CM-H2DCFDA) mixed isomers special packaging 20 x 50 microgC2944 coelenterazine 250 microgD399 2acute7acute-dichlorodihydrouorescein diacetate (2acute7acute-dichlorouorescin diacetate H2DCFDA) 100 mgD2935 2acute7acute-dichlorodihydrouorescein diacetate succinimidyl ester (OxyBURSTreg Green H2DCFDA SE) 5 mgD3861 44-diuoro-5-(4-phenyl-13-butadienyl)-4-bora-3a4a-diaza-s-indacene-3-undecanoic acid (BODIPYreg 581591 C11) 1 mgD23805 dihydrocalcein AM special packaging 20 x 50 microgD1168 dihydroethidium (hydroethidine) 25 mgD23107 dihydroethidium (hydroethidine) 5 mM stabilized solution in DMSO 1 mLD11347 dihydroethidium (hydroethidine) special packaging 10 x 1 mgD632 dihydrorhodamine 123 10 mgD23806 dihydrorhodamine 123 5 mM stabilized solution in DMSO 1 mLD633 dihydrorhodamine 6G 25 mgD7894 diphenyl-1-pyrenylphosphine (DPPP) 5 mgE33856 EnzChekreg Myeloperoxidase (MPO) Activity Assay Kit 400 assays for myeloperoxidase chlorination and peroxidation activity 1 kitF2902 Fc OxyBURSTreg Green assay reagent 25 assays 3 mgmL 500 microLG36000 glutathione ethyl ester biotin amide (BioGEE) glutathiolation detection reagent special packaging 10 x 100 microgH36004 3acute-(p-hydroxyphenyl) uorescein (HPF) 5 mM solution in DMF 470 microLH7476 hypericin 1 mgI36007 Image-iTreg LIVE Green Reactive Oxygen Species Detection Kit for microscopy 1 kitL6868 lucigenin (bis-N-methylacridinium nitrate) high purity 10 mgL8455 luminol (3-aminophthalhydrazide) 25 gM689 malachite green isothiocyanate 10 mgM7913 trans-1-(2acute-methoxyvinyl)pyrene 1 mgM23800 2-methyl-6-(4-methoxyphenyl)-37-dihydroimidazo12-apyrazin-3-one hydrochloride (MCLA) 5 mgM24571 merocyanine 540 25 mgM36008 MitoSOXtrade Red mitochondrial superoxide indicator for live-cell imaging 10 x 50 microgM7511 MitoTrackerreg Orange CM-H2TMRos special packaging 20 x 50 microgM7513 MitoTrackerreg Red CM-H2XRos special packaging 20 x 50 microgM6494 MTT (3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide) 1 gN6495 nitro blue tetrazolium chloride (NBT) 1 gO13291 OxyBURSTreg Green H2HFF BSA special packaging 5 x 1 mgP36005 cis-parinaric acid 3 mM in ethanol 10 mLP6879 N-(110-phenanthrolin-5-yl)iodoacetamide 5 mgP800 B-phycoerythrin 4 mgmL 05 mLP801 R-phycoerythrin 4 mgmL 05 mLR14060 RedoxSensortrade Red CC-1 special packaging 10 x 50 microgR14000 rose bengal diacetate 5 mgS36002 Singlet Oxygen Sensor Green special packaging 10 x 100 microgX6493 XTT (23-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) 100 mgZ33857 Zentrade Myeloperoxidase (MPO) ELISA Kit 200 assays 1 kit







Section 183 Probes for Nitric Oxide Research

183 Probes for Nitric Oxide ResearchNitric oxide (NO) plays a critical role as a molecular mediator of a

variety of physiological processes including blood-pressure regulation and neurotransmission1ndash8 In endothelial cells as well as in neurons and astrocytes NO is synthesized from L-arginine in a reaction catalyzed by nitric oxide synthase (NOS) 9ndash12 (Figure 1831) NO that diuses into smooth muscle cells binds to the heme group of guanylate cyclase

Because free NO is a transient species with a half-life of about 5 seconds many investigations of this gaseous molecule have relied largely on studies of NOS Preparing NO solutions and detecting NO in experimental systems require special precautions to achieve repro-ducibility1314 NO also reacts at diusion-controlled rates with super-oxide to form a strong oxidant peroxynitrite anion 15 (ONOOndash Table 181) Peroxynitrite is a well-known inammatory mediator in various cardiovascular pathologies but has more recently been recognized as a modulator of signal transduction pathways due to its ability to nitrate tyrosine residues and thereby inuence cellular processes dependent on tyrosine phosphorylation1516 Activated macrophages and neutrophils produce nitric oxide and superoxide and thus peroxynitrite anion at similar rates17 NO generators are also reported to produce an accu-mulation of chelatable Zn2+ in hippocampal neuronal perikarya as de-termined with some of our Zn2+ indicators 18 (Section 197 Table 196)

Spontaneous Nitric Oxide Donors and AntagonistSpermine NONOate

Spermine NONOate (S7916) solids provide a means of preparing aqueous NO solutions19 When dissolved in buer cell culture medium or blood spermine NONOate dissociates to form two molecules of NO and one molecule of the corresponding amine 20 (Figure 1832) e delivery of NO can be easily controlled by preparing moderately ba-sic solutions of this NONOate and then lowering the pH to initiate NO generation Spermine NONOate releases NO slowly (half-life of 39 minutes at 37degC in pH 74 buer) making it suitable for whole ani-mal infusions and experiments with long incubations21 as well as for in situ calibration of DAF-FM 22

SNAP and SIN-1NO donors SNAP (S-nitroso-N-acetylpenicillamine N7892

N7927) and SIN-1 (3-morpholinosydnonimine hydrochloride M7891 M7926) spontaneously release NO (and superoxide in the case of SIN-1) under physiological conditions (Figure 1832) thereby stimulating cyclic GMP production23ndash27 SNAP and SIN-1 have been shown to be potent vasodilators in vivo and in vitro and to inhibit smooth muscle cell mitogenesis and proliferation28ndash31 e relation-ship between NO generated from SNAP and SIN-1 and intracellular Ca2+ has been studied using uorescent Ca2+ indicators 32ndash34 (Chapter 19) It has also been reported that NO released from SNAP stimulates Ca2+-independent synaptic vesicle release34 which can be detected with FMreg 1ndash43 (T3163 T35356 Section 161) We oer SNAP and SIN-1 in 25 mg vials (N7892 M7891) and because of their potential instability in solution as specially packaged sets of 20 vials each con-taining 1 mg (N7927 M7926)

Carboxy-PTIO A Nitric Oxide AntagonistCarboxy-PTIO (C7912) is a water-soluble and stable free radical

molecule that reacts stoichiometrically with NO35ndash37 Carboxy-PTIO can be used in vivo to inhibit the physiological eects mediated byNO 353738 or to quantitate NO levels in vitro by ESR spectrometry3940

SNAP A Photoactivatable Nitric Oxide Donor

SNAP (S-nitroso-N-acetylpenicillamine N7892 N7927) has been shown to release nitric oxide (NO) in response to light stimulation in both aqueous and isopropyl alcohol solutions41 e potential spatial and temporal control of NO release made possible by photolysis of NO precursors makes this an attractive approach for generating NO in ex-perimental systems

Detecting Nitric Oxide Nitrite and Nitratee nitric oxide (NO) radical is short-lived and physiological

concentrations are very low42 making in situ detection a challenging proposition NO is readily oxidized to the nitrosonium cation (NO+) which is moderately stable in aqueous solutions but highly reactive with nucleophiles or other nitrogen oxides Under aerobic conditions these reactive nitrogen oxides (Table 181) can be trapped by various amines in particular by aromatic amines to form diazonium salts or by aro-matic 12-diamines to form benzotriazoles (Figure 1833)

DAF-FM Nitric Oxide IndicatorFirst described in 199843 vicinal diamine derivatives of uorescein

generate stronger uorescence signals at longer wavelengths than pro-totypes such as 23-diaminonaphthalene ese characteristics result in much enhanced performance for in situ nitric oxide detection DAF-FM (4-amino-5-methylamino-2 7 -diuorouorescein) is the foremost example of this class of compounds44 We oer DAF-FM (D23841) and its cell-permeant diacetate derivative (D23842 D23844) Like dihydro-uoresein dihydrorhodamine and dihydroethidium probes (Section 182) and in contrast to BAPTA-based Ca2+ indicators (Section 192 Section 193) DAF-FM is an endpoint dosimeter DAF-FM is not a re-versible equilibrium sensor limiting its ability to track rapid uctua-tions of the target analyte (NO) in real time Extracellularly applied DAF-FM diacetate spontaneously crosses the plasma membrane and is cleaved by esterases to generate intracellular DAF-FM which is then oxidized by NO to a triazole product accompanied by increased uo-rescence (Figure 1833 Figure 1834) e uorescence quantum yield of DAF-FM is reported to be 0005 but increases about 160-fold to 081 aer reacting with NO44 e second step of the process as depicted in Figure 1833 is an oversimplication In fact DAF-FM must rst be nonspecically oxidized to an anilinyl radical which then reacts with NO to form the uorescent triazole product45 is mechanistic complication must be borne in mind when interpreting experimental data Specically the question of whether nonspecic pre-oxidation or reaction with NO is the dominant factor controlling observed DAF-FM







Figure 1831 Nitric oxide synthase production of nitric oxide (NO) and L-citrulline from L-arginine and dioxygen (O2)

NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+

L-Arginine L-Citrulline

Figure 1832 Mechanisms of spontaneous NO release by A) Spermine NONOate (S7916) B) SNAP (N7892 N7927) and C) SIN-1 (M7891 M7926)

+ OH_

O2 O2_

O N NN

CH2CNO

+O N N CHCN + NO + H

+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+

2 R S N O RSSR + 2 NO

C

A

B

Figure 1833 Reaction scheme for the detection of nitric oxide (NO) by DAF-FM (D23841) and DAF-FM diacetate (D23842 D23844)

Esterase

DAF-FM diacetate(Nonuorescent cell-permeant)

Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F

DAF-FM(Weakly uorescent)

O

C O

O

OO

NH2NHCH3

F F

Benzotriazole derivative(Fluorescent)

NO

O

C O

O

OO

FF

N

NNH3C

uorescence signals requires critical scrutiny45ndash47 Applications of DAF-FM and DAF-FM diac-etate include

bull Assessment of NO production in transaldolase-decient lymphoblasts by ow cytometry 48

bull Detection of NO accumulation in embryonic cortical neurons following neurotrophin stimulation 49

bull In vivo imaging of NO in zebrash 50

bull Intravital microscopic detection of NO generation associated with angiogenesis in mice 51

bull Quantitation of ATP-induced NO release in rabbit platelets 22

23-DiaminonaphthaleneIn a reaction similar to that of DAF-FM (Figure 1833) 23-diaminonaphthalene (D7918

Figure 1835) reacts with the nitrosonium cation that forms spontaneously from NO to form the uorescent product 1H-naphthotriazole5253 Using 23-diaminonaphthalene researchers have developed a rapid quantitative uorometric assay that can detect from 10 nM to 10 microM nitrite and is compatible with a 96-well microplate format54

Figure 1834 Fluorescence emission spectra of DAF-FM (D23841 D23842) in solutions containing 0 to 12 microM nitric oxide (NO)

Fluo

resc

ence

em

issi

on

Wavelength (nm)500 525 550 600

12 microM NO

11

475 575

089

071

054

036

0180

Figure 1835 23-diaminonaphthalene (D7918)

NH2

NH2








12-DiaminoanthraquinoneFor directly detecting NO levels in vivo we oer 12-diaminoanthraquinone (DAA

D23840) is nitric oxide probe is reported to be nonuorescent until it reacts with NO to pro-duce a red-uorescent precipitate 12-Diaminoanthraquinone has been used to detect changes in NO levels in rat retinas aer injury to the optic nerve55 is methodology may make it possible to test the actions of NO in neurodegeneration inammation and other biological processes e role of NO production in hippocampal long-term potentiation has also been investigated using 12-diaminoanthraquinone for spatial imaging of NO in rat brain slices5657

NBD MethylhydrazineNBD methylhydrazine (N-methyl-4-hydrazino-7-nitrobenzofurazan M20490) is a unique

reagent for the detection of nitrite Reaction of NBD methylhydrazine with NO2ndash in the presence

of mineral acids leads to formation of uorescent products with excitationemission maxima of ~468537 nm is reaction serves as the principle behind a selective uorogenic method for the determination of NO2

ndash (Figure 1836) Although NBD methylhydrazine has been used to quantitate nitrite in water using a uorescence microplate reader58 it does not seem to have been used yet to detect nitrite formed by spontaneous oxidation of NO

Dichlorodihydrouorescein Diacetate and Dihydrorhodamine 123In addition to their extensive use for detecting other reactive oxygen species such as su-

peroxide dichlorodihydrouorescein diacetate (H2DCFDA) and dihydrorhodamine 123 (D399 D632 Section 182) have been reported to be useful for detecting peroxynitrite formation both in solution and in live cells59

Anti-Nitrotyrosine AntibodyHigh levels of nitrotyrosine are associated with a large number of diseases including mul-

tiple sclerosis Alzheimer disease and Parkinson disease60ndash62 Increased levels of nitrotyrosine are also indicative of vascular and tissue injury from ischemiandashreperfusion and inammation61 Several pathways for the nitration of tyrosine have been suggested Peroxynitrite (OONOndash) formed by spontaneous reaction of nitric oxide (NO) with superoxide (bullO2

ndash) elicits downstream tyrosine nitration6163 Heme peroxidases such as myeloperoxidase and eosinophil peroxidase have been shown to utilize hydrogen peroxide (H2O2) to oxidize nitrite (NO2

ndash) and catalyze tyrosine nitration64 In addition other heme proteins such as hemoglobin and catalase may con-tribute to tyrosine nitration using NO as a substrate65 Tryptophan residues can also be oxidized by peroxynitrite66

We oer a high-activity rabbit polyclonal anti-nitrotyrosine antibody (A21285) for detecting nitrotyrosine-containing proteins and peptides is antibody is suitable for both immunohis-tochemical (Figure 1837) and western blotting (Figure 1838) applications and is useful for identifying nitrated proteins and determining the level of protein nitrosylation in tissues6768 Fluorescence of Green Fluorescent Protein (GFP) is extremely sensitive to tyrosine nitration as conrmed by correlated anti-nitrotyrosine immunoreactivity69

S-Nitrosothiol DetectionS-nitrosylation of thiols principally in the form of cysteine sidechains or glutathione is a

primary mechanism for downstream propagation of nitric oxide release events is reversible posttranslational modication regulates enzymatic activity subcellular localization chromatin remodeling and protein degradation4970 e primary reactive nitrogen species responsible for

Figure 1838 Specicity of our rabbit anti-nitrotyrosine an-tibody (A21285) to nitrated proteins Equal amounts of avi-din (A887 lane 1) and CaptAvidintrade biotin-binding protein (C21385 lane 2) were run on an SDS-polyacrylamide gel (4ndash20) and blotted onto a PVDF membrane CaptAvidintrade biotin-binding protein a derivative of avidin has nitrated tyrosine residues in the biotin-binding site On a western blot nitrated proteins were identied with the anti-nitroty-rosine antibody in combination with an alkaline phospha-tase conjugate of goat antindashrabbit IgG antibody (G21079) and the red-uorescent substrate DDAO phosphate (D6487)

CaptAvidintrade Dimer

CaptAvidintrade

1 2

Figure 1836 Reaction scheme illustrating the principle of nitrite detection by NBD methylhydrazine (M20490)

CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+

Figure 1837 Fixed and permeabilized bovine pulmonary artery endothelial cells were treated with either degraded peroxynitrite (top panel) or 100 microM peroxynitrite (bottom panel) for 5 minutes at room temperature to induce protein nitration Nitrated tyrosine residues were detected with our rabbit anti-nitrotyrosine antibody (A21285) and visualized with the green-uorescent Alexa Fluorreg 488 goat antindashrab-bit IgG antibody (A11008) Nuclei were counterstained with blue-uorescent DAPI (D1306 D3571 D21490)







Figure 1839 Principle of nitrite quantitation using the Griess Reagent Kit (G7921) Formation of the azo dye is detected via its absorbance at 548 nm

+HO3S NH2 NH(CH2)2NH2

Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye

S-nitrosylation of protein thiols is dinitrogen dioxide (N2O3) formed from O2 and NO Techniques for detecting S-nitrosothiol modica-tions exploit but are also compromised by their reversible nature and their susceptibility to photolytic cleavage e technique with most widespread adoption oen referred to as the biotin switch method71ndash73 consists of three steps (1) blocking of free thiols with N-ethylmaleimide or another alkylating reagent (2) selective reduction of S-nitrosothiols to thiols using ascorbate or TCEP (T2556 Section 21) and (3) labeling of thiols created in step 2 with a uorescent or biotinylated maleimide or iodoacetamide reagent 7475 (Section 22 or Section 42 respective-ly) Streptavidin agarose (S951 Section 76) can be used to subsequently pull down biotinylated proteins for further analysis if required e over-all technique is vulnerable to false positives through incomplete block-ing of unmodied thiols in step 1 and inadvertent reduction of disuldes in step 2 Other methods take advantage of the fact that S-nitrosothiols can be cleaved by heavy metal ions such as Hg2+ or by exposure to ul-traviolet light releasing NO and subsequently nitrite (NO2

ndash)76 e NO product of this process can be detected using DAF-FM 77 or the nascent thiol product can be detected using a uorescent maleimide reagent78

Griess Reagent KitUnder physiological conditions NO is readily oxidized to nitrite

and nitrate or it is trapped by thiols as an S-nitroso adduct e Griess reagent provides a simple and well characterized colorimetric assay for nitrites and nitrates that have been reduced to nitrites with a detection limit of about 100 nM527980 Nitrites react with sulfanilic acid in acidic solution to form an intermediate diazonium salt that couples to N-(1-naphthyl)ethylenediamine to yield a purple azo derivative that can be monitored by absorbance at 548 nm (Figure 1839)

Our Griess Reagent Kit (G7921) contains all of the reagents re-quired for nitrite quantitation including

bull N-(1-Naphthyl)ethylenediamine dihydrochloridebull Sulfanilic acid in 5 H3PO4bull Concentrated nitrite quantitation standard for generating calibra-

tion curvesbull Detailed protocols for spectrophotometer and microplate reader

assays

Both the N-(1-naphthyl)ethylenediamine dihydrochloride and the sulfanilic acid in 5 H3PO4 are provided in convenient dropper bottles for easy preparation of the Griess reagent Sample pretreatment with nitrate reductase and glucose 6-phosphate dehydrogenase is reported to reduce nitrate without producing excess NADPH which can inter-fere with the Griess reaction81 A review of the use of the Griess re-agent for nitrite and nitrate quantitation in human plasma describes optimal reaction conditions for minimizing interference from plasma constituents (particularly NADPH)82 e Griess Reagent Kit can also

Figure 18310 Linearity and sensitivity of the Measure-iTtrade high-sensitivity nitrite as-say Triplicate 10 microL samples of nitrite were assayed using the Measure-iTtrade High-Sensitivity Nitrite Assay Kit (M36051) Fluorescence was measured using excitationemission of 365450 nm and plotted versus picomoles of nitrite Background uorescence was not sub-tracted The variation (CV) of replicate samples was lt2

Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995

be used to analyze NO that has been trapped as an S-nitroso derivative by a modication that uses mercuric chloride or copper (II) acetate to release the NO from its complex8384

Measure-iTtrade High-Sensitivity Nitrite Assay Kite Measure-iTtrade High-Sensitivity Nitrite Assay Kit (M36051) pro-

vides an easy and accurate method for quantitating nitrite is kit has an optimal range of 20ndash500 picomoles nitrite (Figure 18310) making it up to 50 times more sensitive than colorimetric methods utilizing the Griess reagent Nitrates may be analyzed aer quantitative conver-sion to nitrites through enzymatic reduction 80 used in this manner the Measure-iTtrade nitrite assay also provides an eective method for quan-titating nitric oxide

Each Measure-iTtrade High-Sensitivity Nitrite Assay Kit contains

bull Measure-iTtrade nitrite quantitation reagent (100X concentrate in 062 M HCl)

bull Measure-iTtrade nitrite quantitation developer (28 M NaOH)bull Measure-iTtrade nitrite quantitation standard (11 mM sodium nitrite)bull Detailed protocols

Simply dilute the reagent 1100 load 100 microL into the wells of a microplate add 1ndash10 microL sample volumes and mix Aer a 10-minute incubation at room temperature add 5 microL of developer and read the uorescence e assay signal is stable for at least 3 hours and com-mon contaminants are well tolerated in the assay e Measure-iTtrade High-Sensitivity Nitrite Assay Kit provides sucient material for 2000 assays based on a 100 microL assay volume in a 96-well microplate format this nitrite assay can also be adapted for use in cuvettes or 384-well microplates








1 Science (2001) 2922413 2 Science (2001) 2922486 3 Curr Biol (1997) 7R376 4 Cell (1994) 78919 5 Am J Physiol (1992) 262G379 6 Annu Rev Biochem (1994) 63175 7 J Med Chem (1995) 384343 8 Science (1992) 257494 9 Biochem J (2001) 357593 10 Biochem J (1994) 298249 11 Cell (1994) 78915 12 J Med Chem (1994) 371899 13 Methods Mol Biol (1998) 100215 14 Curr Opin Chem Biol (2010) 1443 15 Free Radic Biol Med (2001) 30463 16 Front Biosci (2009) 144809 17 Methods Enzymol (1994) 233229 18 Brain Res (1998) 799118 19 Methods Enzymol (1996) 268281 20 J Med Chem (1991) 343242 21 J Biol Chem (2001) 27628799 22 Anal Chem (2007) 792421 23 Nature (1993) 364626 24 Nature (1995) 37568 25 Brain Res (1993) 619344 26 FEBS Lett (1993) 315139 27 romb Res (1993) 70405 28 J Pharmacol Exp er (1992) 260286 29 Eur J Pharmacol (1987) 144379 30 J Pharmacol Exp er (1989) 248762 31 J Clin Invest (1989) 831774 32 Am J Physiol (1994) 266L9 33 Life Sci (1994) 541449 34 Neuron (1994) 121235 35 Biochemistry (1995) 347177 36 Biochem Biophys Res Commun (1994) 202923 37 Biochemistry (1993) 32827 38 Infect Immun (1993) 613552 39 Photochem Photobiol (1995) 61325 40 Life Sci (1994) 54185 41 Photochem Photobiol (1998) 67282 42 Nitric Oxide (2009) 2192 43 Anal Chem (1998) 702446 44 Angew Chem Int Ed Engl (1999) 383209 45 Free

DATA TABLE 183 PROBES FOR NITRIC OXIDE RESEARCHCat No MW Storage Soluble Abs EC Em Solvent NotesC7912 31539 FFD H2O 367 9300 none MeOHD7915 15513 FFDDA H2O DMSO 248 8000 none pH 12 1D7918 15820 L DMSO MeOH 340 5100 377 MeOH 2D23840 33632 FDL DMSO 521 6000 none MeOH 3D23841 41235 FDL DMSO 487 84000 see Notes pH 8 4D23842 49642 FDL DMSO lt300 none 5D23844 49642 FDL DMSO lt300 none 5M7891 20663 FFDLL DMSO H2O 291 11000 none pH 7 6M7926 20663 FFDLL DMSO H2O 291 11000 none pH 7 6M20490 20916 FL MeCN 487 24000 none MeOH 7N7892 22024 FFDLL DMSO H2O 342 700 none MeOH 6N7927 22024 FFDLL DMSO H2O 342 700 none MeOH 6S7916 26235 FFDDA H2O DMSO 248 8200 none pH 12 1For denitions of the contents of this data table see ldquoUsing The Molecular Probesreg Handbookrdquo in the introductory pages

Notes1 Releases nitric oxide upon acid-catalyzed dissociation in solution Stable in alkaline solutions (Methods Enzymol (1996) 268281)2 Fluorescence of D7918 is weak Reaction with nitrite yields highly uorescent 1H-naphthotriazole (Abs = 365 nm Em = 415 nm in H2O (pH 12)) (Methods Enzymol (1996) 268105)3 12-Diaminoanthraquinone reacts with nitrite or nitric oxide to produce 1H-anthratriazole-611-dione which forms a red-uorescent (Em gt580 nm) precipitate in water (Neuroreport (1998) 94051)4 DAF-FM uorescence is very weak Reaction with nitrite or nitric oxide generates a highly uorescent benzotriazole derivative with Abs = 495 nm (EC = 73000 cmndash1Mndash1) Em = 515 nm in pH 74

buer (Angew Chem Int Ed Engl (1999) 383209)5 Acetate hydrolysis and subsequent reaction with nitrite or nitric oxide generate a highly uorescent benzotriazole derivative with Abs = 495 nm (EC = 73000 cmndash1Mndash1) Em = 515 nm in pH 74

buer (Angew Chem Int Ed Engl (1999) 383209)6 Spontaneously decomposes in solution7 NBD methylhydrazine reacts with nitrite in the presence of strong acid to form uorescent N-methyl-4-amino-7-nitrobenzofurazan (Abs = 459 nm Em = 537 nm in MeCN) (Anal Chem (1999)

713003)

PRODUCT LIST 183 PROBES FOR NITRIC OXIDE RESEARCHCat No Product QuantityA21285 anti-nitrotyrosine rabbit IgG fraction 1 mgmL 05 mLC7912 2-(4-carboxyphenyl)-4455-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (carboxy-PTIO) 25 mgD23841 DAF-FM (4-amino-5-methylamino-2acute7acute-diuorouorescein) 1 mgD23842 DAF-FM diacetate (4-amino-5-methylamino-2acute7acute-diuorouorescein diacetate) 1 mgD23844 DAF-FM diacetate (4-amino-5-methylamino-2acute7acute-diuorouorescein diacetate) special packaging 10 x 50 microgD23840 12-diaminoanthraquinone sulfate (DAA) high purity 5 mgD7918 23-diaminonaphthalene 100 mgG7921 Griess Reagent Kit for nitrite quantitation 1 kitM36051 Measure-iTtrade High-Sensitivity Nitrite Assay Kit 2000 assays 1 kitM20490 N-methyl-4-hydrazino-7-nitrobenzofurazan (NBD methylhydrazine) 25 mgM7891 3-morpholinosydnonimine hydrochloride (SIN-1) 25 mgM7926 3-morpholinosydnonimine hydrochloride (SIN-1) special packaging 20 x 1 mgN7892 S-nitroso-N-acetylpenicillamine (SNAP) 25 mgN7927 S-nitroso-N-acetylpenicillamine (SNAP) special packaging 20 x 1 mgS7916 spermine NONOate 10 mg

REFERENCESRadic Biol Med (2007) 43995 46 Anal Chem (2006) 781859 47 Free Radic Biol Med (2005) 39327 48 Biochem J (2008) 415123 49 Nature (2008) 455411 50 Free Radic Biol Med (2007) 43619 51 Methods Enzymol (2008) 441393 52 Luminescence (1999) 14283 53 Methods Enzymol (1996) 268105 54 Anal Biochem (1993) 21411 55 Neuroreport (1998) 94051 56 Neurobiol Dis (2002) 1196 57 Neuroimage (2002) 15633 58 Anal Chem (1999) 713003 59 Methods Enzymol (2008) 441261 60 J Biol Chem (2003) 2788380 61 Free Radic Res (2000) 33771 62 Methods Enzymol (1999) 301373 63 Proc Natl Acad Sci U S A (1996) 9311853 64 Biochem Biophys Res Commun (2001) 285273 65 Biochim Biophys Acta (2001) 152897 66 Biochem Biophys Res Commun (1997) 23482 67 Nat Clin Pract Cardiovasc Med (2008) 5811 68 Methods Mol Biol (2008) 47741 69 Proc Natl Acad Sci U S A (2002) 993481 70 Proc Natl Acad Sci U S A (2005) 102117 71 Free Radic Biol Med (2009) 46119 72 Proteomics (2009) 9808 73 Methods Enzymol (2008) 44153 74 Nitric Oxide (2008) 19295 75 J Bacteriol (2008) 1904997 76 J Biol Chem (1996) 27118596 77 Anal Biochem (2005) 34669 78 J Biol Chem (2006) 28140354 79 FASEB J (1993) 7349 80 Anal Biochem (1982) 126131 81 Anal Biochem (1995) 224502 82 Methods Enzymol (2008) 440361 83 Anal Biochem (1996) 238150 84 Chem Biol (1996) 3655





EIG

HTE

EN

CHAPTER 18

Probes for Reactive Oxygen Species Including Nitric Oxide181 Introduction to Reactive Oxygen Species 805

182 Generating and Detecting Reactive Oxygen Species 806

Generating Singlet Oxygen 806

Hypericin 806

Rose Bengal Diacetate 806

Merocyanine 540 806

Detecting Singlet Oxygen 807

Singlet Oxygen Sensor Green Reagent 807

trans-1-(2acute-Methoxyvinyl)pyrene 807

Generating Hydroxyl and Superoxide Radicals 807

Malachite Green 807

110-Phenanthroline Iodoacetamide 807

Detecting Hydroxyl and Superoxide Radicals 808

MitoSOXtrade Red Mitochondrial Superoxide Indicator 808

Dihydroethidium (Hydroethidine) 808

Fluorogenic Spin Traps 809

Chemiluminescent and Chromogenic Reagents for Detecting Superoxide 809

Detecting Peroxides Peroxyl Radicals and Lipid Peroxidation 810

cis-Parinaric Acid 810

Diphenyl-1-Pyrenylphosphine 810

BODIPYreg 581591 C A Ratiometric Lipid Peroxidation Sensor 811

Other Scavengers for Peroxyl Radicals 811

Luminol 811

Detecting 4-Hydroxy-2-Nonenal 811

Detecting Peroxides and Peroxidases with Amplexreg Red Reagents 811

Amplexreg Red Reagent Stable Substrate for Peroxidase Detection 811

Amplexreg UltraRed Reagent Brighter and More Sensitive than the Amplexreg Red Reagent 812

Amplexreg Red Hydrogen PeroxidePeroxidase Assay Kit 812

Amplexreg Red XanthineXanthine Oxidase Assay Kit 812

EnzChekreg Myeloperoxidase (MPO) Activity Assay Kit 813

Zentrade Myeloperoxidase (MPO) ELISA Kit 814

Assaying Oxidative Activity in Live Cells 815

Dichlorodihydrouorescein Diacetate 815

Improved Versions of H2DCFDA 816

Image-iTreg LIVE Green Reactive Oxygen Species Detection Kit 817

Aminophenyl Fluorescein and Hydroxyphenyl Fluorescein 817

Dihydrocalcein AM 818

OxyBURSTreg Green Reagents 818

Amine-Reactive OxyBURSTreg Green Reagent 818



thermofi shercomprobes















SNAP and SIN-1 824


































































bull MCLA (M23800) 6566

bull MTT (M6494) 67

bull NBT (N6495) 68




























0

Time (sec)

300100 200

2000

1000

0

Fluo

resc

ence

50 D2O

Tris buffer

NaN3

0

Time (sec)

300100 200

300

200

100

0

Fluo

resc

ence




A

B








O

O

OH

OHOH

OHOH

OH

H3CH3C















C CH OCH3

H1O2

C

O O

CH H

OCH3

CL (Em = 465 nm)

CH

O


C

N(CH3)2(CH3)2N

N C S

ClO4


N N

NH C CH2I

O
















H H

HCHCH3


ndash)

N

(CH2)6 P

H2N NH2

H

3

N

(CH2)6 P

H2N NH2

3

OH

O2


1

10

100

1000

10000

Super

oxide

Super

oxide +

SOD

No ad

ditionH 2

O 2

H 2O 2

+ H

RP

Singlet

oxy

gen

Perox

ynitr

ite

Super

oxide

Super

oxide +

SOD

Super

oxide (

no D

NA)

No ad

dition

H 2O 2

H 2O 2

+ H

RP

Nitric

oxide

Singlet

oxy

gen

Perox

ynitr

ite

Rel

ativ

e u

ores

cenc

e

0

02

04

06

08

10

12

14

16

Sodiumnitrite

Nitricoxide

Peroxy-nitrite

Ab

sorb

ance

(548

nm

)


























N

C

NH

O

N

O

CH3

CH3

H3C

H3C


N

O

CH3

CH3

H3C

H3C

NCH2

OHO C

O

KO


CH3OH

OCH3 HC









































(CH2)7CCCC

CCCC

H

CH3CH2

H H

H

H

H

H

H

C OH

O









N

O OHHO

C CH3

O



Glucose Oxidase

H2O2 O2

N

O OHHO

C CH3O

N

O OHO

OHO

HO

CH2OH

O

OH

OHO

HO

CH2OHOH

OH
































30201000

100

500

400

300

200

0 200 400 600 800 1000


0

10000

5000

15000

20000

Fluo

resc

ence

Amplexreg

Red

Amplexreg

UltraRed


1 2 3 4 5 6 7 8 9 10

pH

Fluo

resc

ence

em

issi

on

Amplexreg Red

Amplexreg UltraRed


Wavelength (nm)

Ab

sorp

tion

Fluo

resc

ence

em

issi

on

400 650600450 500 550 700








HRP (mUmL)

Fluo

resc

ence

00

500

1000

1500

2000

2500

3000

3500

1 2 3 4 5 6 7

0

100

200

300

400

500

0 01 02 03 04





























HOCl Clndash

H2O2

AH2

AH2A

AH2

AH

AH

AH

AH+ +

MPO-II




0 1601208040 200

1400

0

2800

4200

5600

7000

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 963 120

100

200

00

40 80 120 160 200

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

4000

3200

2400

1600

800

0 129630

100

200

A

B

















MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)



















SNAP and SIN-1 824


































































bull MCLA (M23800) 6566

bull MTT (M6494) 67

bull NBT (N6495) 68




























0

Time (sec)

300100 200

2000

1000

0

Fluo

resc

ence

50 D2O

Tris buffer

NaN3

0

Time (sec)

300100 200

300

200

100

0

Fluo

resc

ence




A

B








O

O

OH

OHOH

OHOH

OH

H3CH3C















C CH OCH3

H1O2

C

O O

CH H

OCH3

CL (Em = 465 nm)

CH

O


C

N(CH3)2(CH3)2N

N C S

ClO4


N N

NH C CH2I

O
















H H

HCHCH3


ndash)

N

(CH2)6 P

H2N NH2

H

3

N

(CH2)6 P

H2N NH2

3

OH

O2


1

10

100

1000

10000

Super

oxide

Super

oxide +

SOD

No ad

ditionH 2

O 2

H 2O 2

+ H

RP

Singlet

oxy

gen

Perox

ynitr

ite

Super

oxide

Super

oxide +

SOD

Super

oxide (

no D

NA)

No ad

dition

H 2O 2

H 2O 2

+ H

RP

Nitric

oxide

Singlet

oxy

gen

Perox

ynitr

ite

Rel

ativ

e u

ores

cenc

e

0

02

04

06

08

10

12

14

16

Sodiumnitrite

Nitricoxide

Peroxy-nitrite

Ab

sorb

ance

(548

nm

)


























N

C

NH

O

N

O

CH3

CH3

H3C

H3C


N

O

CH3

CH3

H3C

H3C

NCH2

OHO C

O

KO


CH3OH

OCH3 HC









































(CH2)7CCCC

CCCC

H

CH3CH2

H H

H

H

H

H

H

C OH

O









N

O OHHO

C CH3

O



Glucose Oxidase

H2O2 O2

N

O OHHO

C CH3O

N

O OHO

OHO

HO

CH2OH

O

OH

OHO

HO

CH2OHOH

OH
































30201000

100

500

400

300

200

0 200 400 600 800 1000


0

10000

5000

15000

20000

Fluo

resc

ence

Amplexreg

Red

Amplexreg

UltraRed


1 2 3 4 5 6 7 8 9 10

pH

Fluo

resc

ence

em

issi

on

Amplexreg Red

Amplexreg UltraRed


Wavelength (nm)

Ab

sorp

tion

Fluo

resc

ence

em

issi

on

400 650600450 500 550 700








HRP (mUmL)

Fluo

resc

ence

00

500

1000

1500

2000

2500

3000

3500

1 2 3 4 5 6 7

0

100

200

300

400

500

0 01 02 03 04





























HOCl Clndash

H2O2

AH2

AH2A

AH2

AH

AH

AH

AH+ +

MPO-II




0 1601208040 200

1400

0

2800

4200

5600

7000

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 963 120

100

200

00

40 80 120 160 200

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

4000

3200

2400

1600

800

0 129630

100

200

A

B

















MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)






















































bull MCLA (M23800) 6566

bull MTT (M6494) 67

bull NBT (N6495) 68




























0

Time (sec)

300100 200

2000

1000

0

Fluo

resc

ence

50 D2O

Tris buffer

NaN3

0

Time (sec)

300100 200

300

200

100

0

Fluo

resc

ence




A

B








O

O

OH

OHOH

OHOH

OH

H3CH3C















C CH OCH3

H1O2

C

O O

CH H

OCH3

CL (Em = 465 nm)

CH

O


C

N(CH3)2(CH3)2N

N C S

ClO4


N N

NH C CH2I

O
















H H

HCHCH3


ndash)

N

(CH2)6 P

H2N NH2

H

3

N

(CH2)6 P

H2N NH2

3

OH

O2


1

10

100

1000

10000

Super

oxide

Super

oxide +

SOD

No ad

ditionH 2

O 2

H 2O 2

+ H

RP

Singlet

oxy

gen

Perox

ynitr

ite

Super

oxide

Super

oxide +

SOD

Super

oxide (

no D

NA)

No ad

dition

H 2O 2

H 2O 2

+ H

RP

Nitric

oxide

Singlet

oxy

gen

Perox

ynitr

ite

Rel

ativ

e u

ores

cenc

e

0

02

04

06

08

10

12

14

16

Sodiumnitrite

Nitricoxide

Peroxy-nitrite

Ab

sorb

ance

(548

nm

)


























N

C

NH

O

N

O

CH3

CH3

H3C

H3C


N

O

CH3

CH3

H3C

H3C

NCH2

OHO C

O

KO


CH3OH

OCH3 HC









































(CH2)7CCCC

CCCC

H

CH3CH2

H H

H

H

H

H

H

C OH

O









N

O OHHO

C CH3

O



Glucose Oxidase

H2O2 O2

N

O OHHO

C CH3O

N

O OHO

OHO

HO

CH2OH

O

OH

OHO

HO

CH2OHOH

OH
































30201000

100

500

400

300

200

0 200 400 600 800 1000


0

10000

5000

15000

20000

Fluo

resc

ence

Amplexreg

Red

Amplexreg

UltraRed


1 2 3 4 5 6 7 8 9 10

pH

Fluo

resc

ence

em

issi

on

Amplexreg Red

Amplexreg UltraRed


Wavelength (nm)

Ab

sorp

tion

Fluo

resc

ence

em

issi

on

400 650600450 500 550 700








HRP (mUmL)

Fluo

resc

ence

00

500

1000

1500

2000

2500

3000

3500

1 2 3 4 5 6 7

0

100

200

300

400

500

0 01 02 03 04





























HOCl Clndash

H2O2

AH2

AH2A

AH2

AH

AH

AH

AH+ +

MPO-II




0 1601208040 200

1400

0

2800

4200

5600

7000

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 963 120

100

200

00

40 80 120 160 200

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

4000

3200

2400

1600

800

0 129630

100

200

A

B

















MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)























0

Time (sec)

300100 200

2000

1000

0

Fluo

resc

ence

50 D2O

Tris buffer

NaN3

0

Time (sec)

300100 200

300

200

100

0

Fluo

resc

ence




A

B








O

O

OH

OHOH

OHOH

OH

H3CH3C















C CH OCH3

H1O2

C

O O

CH H

OCH3

CL (Em = 465 nm)

CH

O


C

N(CH3)2(CH3)2N

N C S

ClO4


N N

NH C CH2I

O
















H H

HCHCH3


ndash)

N

(CH2)6 P

H2N NH2

H

3

N

(CH2)6 P

H2N NH2

3

OH

O2


1

10

100

1000

10000

Super

oxide

Super

oxide +

SOD

No ad

ditionH 2

O 2

H 2O 2

+ H

RP

Singlet

oxy

gen

Perox

ynitr

ite

Super

oxide

Super

oxide +

SOD

Super

oxide (

no D

NA)

No ad

dition

H 2O 2

H 2O 2

+ H

RP

Nitric

oxide

Singlet

oxy

gen

Perox

ynitr

ite

Rel

ativ

e u

ores

cenc

e

0

02

04

06

08

10

12

14

16

Sodiumnitrite

Nitricoxide

Peroxy-nitrite

Ab

sorb

ance

(548

nm

)


























N

C

NH

O

N

O

CH3

CH3

H3C

H3C


N

O

CH3

CH3

H3C

H3C

NCH2

OHO C

O

KO


CH3OH

OCH3 HC









































(CH2)7CCCC

CCCC

H

CH3CH2

H H

H

H

H

H

H

C OH

O









N

O OHHO

C CH3

O



Glucose Oxidase

H2O2 O2

N

O OHHO

C CH3O

N

O OHO

OHO

HO

CH2OH

O

OH

OHO

HO

CH2OHOH

OH
































30201000

100

500

400

300

200

0 200 400 600 800 1000


0

10000

5000

15000

20000

Fluo

resc

ence

Amplexreg

Red

Amplexreg

UltraRed


1 2 3 4 5 6 7 8 9 10

pH

Fluo

resc

ence

em

issi

on

Amplexreg Red

Amplexreg UltraRed


Wavelength (nm)

Ab

sorp

tion

Fluo

resc

ence

em

issi

on

400 650600450 500 550 700








HRP (mUmL)

Fluo

resc

ence

00

500

1000

1500

2000

2500

3000

3500

1 2 3 4 5 6 7

0

100

200

300

400

500

0 01 02 03 04





























HOCl Clndash

H2O2

AH2

AH2A

AH2

AH

AH

AH

AH+ +

MPO-II




0 1601208040 200

1400

0

2800

4200

5600

7000

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 963 120

100

200

00

40 80 120 160 200

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

4000

3200

2400

1600

800

0 129630

100

200

A

B

















MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)










O

O

OH

OHOH

OHOH

OH

H3CH3C















C CH OCH3

H1O2

C

O O

CH H

OCH3

CL (Em = 465 nm)

CH

O


C

N(CH3)2(CH3)2N

N C S

ClO4


N N

NH C CH2I

O
















H H

HCHCH3


ndash)

N

(CH2)6 P

H2N NH2

H

3

N

(CH2)6 P

H2N NH2

3

OH

O2


1

10

100

1000

10000

Super

oxide

Super

oxide +

SOD

No ad

ditionH 2

O 2

H 2O 2

+ H

RP

Singlet

oxy

gen

Perox

ynitr

ite

Super

oxide

Super

oxide +

SOD

Super

oxide (

no D

NA)

No ad

dition

H 2O 2

H 2O 2

+ H

RP

Nitric

oxide

Singlet

oxy

gen

Perox

ynitr

ite

Rel

ativ

e u

ores

cenc

e

0

02

04

06

08

10

12

14

16

Sodiumnitrite

Nitricoxide

Peroxy-nitrite

Ab

sorb

ance

(548

nm

)


























N

C

NH

O

N

O

CH3

CH3

H3C

H3C


N

O

CH3

CH3

H3C

H3C

NCH2

OHO C

O

KO


CH3OH

OCH3 HC









































(CH2)7CCCC

CCCC

H

CH3CH2

H H

H

H

H

H

H

C OH

O









N

O OHHO

C CH3

O



Glucose Oxidase

H2O2 O2

N

O OHHO

C CH3O

N

O OHO

OHO

HO

CH2OH

O

OH

OHO

HO

CH2OHOH

OH
































30201000

100

500

400

300

200

0 200 400 600 800 1000


0

10000

5000

15000

20000

Fluo

resc

ence

Amplexreg

Red

Amplexreg

UltraRed


1 2 3 4 5 6 7 8 9 10

pH

Fluo

resc

ence

em

issi

on

Amplexreg Red

Amplexreg UltraRed


Wavelength (nm)

Ab

sorp

tion

Fluo

resc

ence

em

issi

on

400 650600450 500 550 700








HRP (mUmL)

Fluo

resc

ence

00

500

1000

1500

2000

2500

3000

3500

1 2 3 4 5 6 7

0

100

200

300

400

500

0 01 02 03 04





























HOCl Clndash

H2O2

AH2

AH2A

AH2

AH

AH

AH

AH+ +

MPO-II




0 1601208040 200

1400

0

2800

4200

5600

7000

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 963 120

100

200

00

40 80 120 160 200

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

4000

3200

2400

1600

800

0 129630

100

200

A

B

















MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)

















H H

HCHCH3


ndash)

N

(CH2)6 P

H2N NH2

H

3

N

(CH2)6 P

H2N NH2

3

OH

O2


1

10

100

1000

10000

Super

oxide

Super

oxide +

SOD

No ad

ditionH 2

O 2

H 2O 2

+ H

RP

Singlet

oxy

gen

Perox

ynitr

ite

Super

oxide

Super

oxide +

SOD

Super

oxide (

no D

NA)

No ad

dition

H 2O 2

H 2O 2

+ H

RP

Nitric

oxide

Singlet

oxy

gen

Perox

ynitr

ite

Rel

ativ

e u

ores

cenc

e

0

02

04

06

08

10

12

14

16

Sodiumnitrite

Nitricoxide

Peroxy-nitrite

Ab

sorb

ance

(548

nm

)


























N

C

NH

O

N

O

CH3

CH3

H3C

H3C


N

O

CH3

CH3

H3C

H3C

NCH2

OHO C

O

KO


CH3OH

OCH3 HC









































(CH2)7CCCC

CCCC

H

CH3CH2

H H

H

H

H

H

H

C OH

O









N

O OHHO

C CH3

O



Glucose Oxidase

H2O2 O2

N

O OHHO

C CH3O

N

O OHO

OHO

HO

CH2OH

O

OH

OHO

HO

CH2OHOH

OH
































30201000

100

500

400

300

200

0 200 400 600 800 1000


0

10000

5000

15000

20000

Fluo

resc

ence

Amplexreg

Red

Amplexreg

UltraRed


1 2 3 4 5 6 7 8 9 10

pH

Fluo

resc

ence

em

issi

on

Amplexreg Red

Amplexreg UltraRed


Wavelength (nm)

Ab

sorp

tion

Fluo

resc

ence

em

issi

on

400 650600450 500 550 700








HRP (mUmL)

Fluo

resc

ence

00

500

1000

1500

2000

2500

3000

3500

1 2 3 4 5 6 7

0

100

200

300

400

500

0 01 02 03 04





























HOCl Clndash

H2O2

AH2

AH2A

AH2

AH

AH

AH

AH+ +

MPO-II




0 1601208040 200

1400

0

2800

4200

5600

7000

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 963 120

100

200

00

40 80 120 160 200

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

4000

3200

2400

1600

800

0 129630

100

200

A

B

















MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)




























N

C

NH

O

N

O

CH3

CH3

H3C

H3C


N

O

CH3

CH3

H3C

H3C

NCH2

OHO C

O

KO


CH3OH

OCH3 HC









































(CH2)7CCCC

CCCC

H

CH3CH2

H H

H

H

H

H

H

C OH

O









N

O OHHO

C CH3

O



Glucose Oxidase

H2O2 O2

N

O OHHO

C CH3O

N

O OHO

OHO

HO

CH2OH

O

OH

OHO

HO

CH2OHOH

OH
































30201000

100

500

400

300

200

0 200 400 600 800 1000


0

10000

5000

15000

20000

Fluo

resc

ence

Amplexreg

Red

Amplexreg

UltraRed


1 2 3 4 5 6 7 8 9 10

pH

Fluo

resc

ence

em

issi

on

Amplexreg Red

Amplexreg UltraRed


Wavelength (nm)

Ab

sorp

tion

Fluo

resc

ence

em

issi

on

400 650600450 500 550 700








HRP (mUmL)

Fluo

resc

ence

00

500

1000

1500

2000

2500

3000

3500

1 2 3 4 5 6 7

0

100

200

300

400

500

0 01 02 03 04





























HOCl Clndash

H2O2

AH2

AH2A

AH2

AH

AH

AH

AH+ +

MPO-II




0 1601208040 200

1400

0

2800

4200

5600

7000

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 963 120

100

200

00

40 80 120 160 200

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

4000

3200

2400

1600

800

0 129630

100

200

A

B

















MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)










































(CH2)7CCCC

CCCC

H

CH3CH2

H H

H

H

H

H

H

C OH

O









N

O OHHO

C CH3

O



Glucose Oxidase

H2O2 O2

N

O OHHO

C CH3O

N

O OHO

OHO

HO

CH2OH

O

OH

OHO

HO

CH2OHOH

OH
































30201000

100

500

400

300

200

0 200 400 600 800 1000


0

10000

5000

15000

20000

Fluo

resc

ence

Amplexreg

Red

Amplexreg

UltraRed


1 2 3 4 5 6 7 8 9 10

pH

Fluo

resc

ence

em

issi

on

Amplexreg Red

Amplexreg UltraRed


Wavelength (nm)

Ab

sorp

tion

Fluo

resc

ence

em

issi

on

400 650600450 500 550 700








HRP (mUmL)

Fluo

resc

ence

00

500

1000

1500

2000

2500

3000

3500

1 2 3 4 5 6 7

0

100

200

300

400

500

0 01 02 03 04





























HOCl Clndash

H2O2

AH2

AH2A

AH2

AH

AH

AH

AH+ +

MPO-II




0 1601208040 200

1400

0

2800

4200

5600

7000

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 963 120

100

200

00

40 80 120 160 200

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

4000

3200

2400

1600

800

0 129630

100

200

A

B

















MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)











N

O OHHO

C CH3

O



Glucose Oxidase

H2O2 O2

N

O OHHO

C CH3O

N

O OHO

OHO

HO

CH2OH

O

OH

OHO

HO

CH2OHOH

OH
































30201000

100

500

400

300

200

0 200 400 600 800 1000


0

10000

5000

15000

20000

Fluo

resc

ence

Amplexreg

Red

Amplexreg

UltraRed


1 2 3 4 5 6 7 8 9 10

pH

Fluo

resc

ence

em

issi

on

Amplexreg Red

Amplexreg UltraRed


Wavelength (nm)

Ab

sorp

tion

Fluo

resc

ence

em

issi

on

400 650600450 500 550 700








HRP (mUmL)

Fluo

resc

ence

00

500

1000

1500

2000

2500

3000

3500

1 2 3 4 5 6 7

0

100

200

300

400

500

0 01 02 03 04





























HOCl Clndash

H2O2

AH2

AH2A

AH2

AH

AH

AH

AH+ +

MPO-II




0 1601208040 200

1400

0

2800

4200

5600

7000

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 963 120

100

200

00

40 80 120 160 200

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

4000

3200

2400

1600

800

0 129630

100

200

A

B

















MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)




















30201000

100

500

400

300

200

0 200 400 600 800 1000


0

10000

5000

15000

20000

Fluo

resc

ence

Amplexreg

Red

Amplexreg

UltraRed


1 2 3 4 5 6 7 8 9 10

pH

Fluo

resc

ence

em

issi

on

Amplexreg Red

Amplexreg UltraRed


Wavelength (nm)

Ab

sorp

tion

Fluo

resc

ence

em

issi

on

400 650600450 500 550 700








HRP (mUmL)

Fluo

resc

ence

00

500

1000

1500

2000

2500

3000

3500

1 2 3 4 5 6 7

0

100

200

300

400

500

0 01 02 03 04





























HOCl Clndash

H2O2

AH2

AH2A

AH2

AH

AH

AH

AH+ +

MPO-II




0 1601208040 200

1400

0

2800

4200

5600

7000

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 963 120

100

200

00

40 80 120 160 200

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

4000

3200

2400

1600

800

0 129630

100

200

A

B

















MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)










HRP (mUmL)

Fluo

resc

ence

00

500

1000

1500

2000

2500

3000

3500

1 2 3 4 5 6 7

0

100

200

300

400

500

0 01 02 03 04





























HOCl Clndash

H2O2

AH2

AH2A

AH2

AH

AH

AH

AH+ +

MPO-II




0 1601208040 200

1400

0

2800

4200

5600

7000

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 963 120

100

200

00

40 80 120 160 200

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

4000

3200

2400

1600

800

0 129630

100

200

A

B

















MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)



















HOCl Clndash

H2O2

AH2

AH2A

AH2

AH

AH

AH

AH+ +

MPO-II




0 1601208040 200

1400

0

2800

4200

5600

7000

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 963 120

100

200

00

40 80 120 160 200

MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

4000

3200

2400

1600

800

0 129630

100

200

A

B

















MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)



















MPO (ngmL)

Rel

ativ

e u

ores

cenc

e

0 10080604020

0 21505 10

500

1000

25 353

3600

2400

1200

0




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)




















O OCCH3

COCH2OCCH3

O O

HCl Cl

CH3COCH2OC

CH3CO

O O

O O



CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO


100 101 102 103 104

Cou

nts

Green uorescence

120

80

40

0



























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)





























COO

OO O

XH

COO

OO O

OX

ROS






















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)





















O OCCH3

CH

C C

CH3COO O

O

O

OO




CH3CO O OCCH3

COCHOCCH3

CH CH


O OO O

OO

H


















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)




















H O H

CO

OCH3

C


(CH3)2N O N(CH3)2

CH2Cl

H


O

CHC

H


CH3 O CH3

H


CHCHCHCHCH

O

H

H

OH

H

H


O O O

CHHCOH

O













CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)














CH3

CH3



REFERENCES





































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)







































































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)



























































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)







































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)































NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)










NH2C NH2NH

C

CHHOOC

NH3

H2)3

+

+

O2

NO Synthase

NADPNADPH

+

H2)3

NH2C O

NH

(C

CHHOOC

NH3

NO+ +)

+



+ OH_

O2 O2_

O N NN

CH2CNO


+ O N N

N O

NH+_

RN N OO

_

RH + 2 NOH+


C

A

B


Esterase


Cell membrane

O

OO

OOCCH3 C CH3

OO

NH2NHCH3

F F


O

C O

O

OO

NH2NHCH3

F F


NO

O

C O

O

OO

FF

N

NNH3C










Fluo

resc

ence

em

issi

on


12 microM NO

11

475 575

089

071

054

036

0180


NH2

NH2

























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)


























CaptAvidintrade

1 2


CH3

NO

N

NO2

N NH2

NO

N

NO2

NHCH3NO2

mdash H

+










Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)











Diazonium salt

NO2_

HO3S N2+

NNHO3S NH(CH2)2NH2

Azo dye








assays



Nitrite (pmol)

0 400300200100 500

Fluo

resc

ence

R2 = 09995




















713003)














713003)





Documents

CHAPTER 1 CHAPTER 18 Fluorophores and Probes for Reactive ... · Free Radic Res (2000) 32:265; 66. Anal Biochem (1999) 271:53; 67. Free Radic Res Commun (1993) 18:369; 68. Arch Biochem