CH 18 Reactive Oxygen Species

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CHAPTER 1
Fluorophores
CHAPTER 18 and
Their for
Probes Amine-Reactive
Reactive
Derivatives
Oxygen Species, Including
Nitric Oxide
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EIGHTEEN
CHAPTER 18
Probes for Reactive Oxygen Species,
Including Nitric Oxide
18.1 Introduction to Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805
18.2 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-(2´-Methoxyvinyl)pyrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807
Generating Hydroxyl and Superoxide Radicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807
Malachite Green. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807
1,10-Phenanthroline Iodoacetamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807
Detecting Hydroxyl and Superoxide Radicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 808
MitoSOX™ 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
BODIPY® 581/591 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 Amplex® Red Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 811
Amplex® Red Reagent: Stable Substrate for Peroxidase Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 811
Amplex® UltraRed Reagent: Brighter and More Sensitive than the Amplex® Red Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 812
Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 812
Amplex® Red Xanthine/Xanthine Oxidase Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 812
EnzChek® Myeloperoxidase (MPO) Activity Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 813
Zen™ Myeloperoxidase (MPO) ELISA Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 814
Assaying Oxidative Activity in Live Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 815
Dichlorodihydrofluorescein Diacetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 815
Improved Versions of H2DCFDA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 816
Image-iT® LIVE Green Reactive Oxygen Species Detection Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 817
Aminophenyl Fluorescein and Hydroxyphenyl Fluorescein. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 817
Dihydrocalcein AM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 818
OxyBURST® Green Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 818
Amine-Reactive OxyBURST® Green Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 818
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Chapter 18 — Probes for Reactive Oxygen Species, Including Nitric Oxide
Dihydrorhodamine 123 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 818

A Longer-Wavelength Reduced Rhodamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819
Reduced MitoTracker® Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819
RedoxSensor™ Red CC-1 Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819
Glutathiolation Detection with BioGEE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819
Tetrazolium Salts: Chromogenic Redox Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 820
Data Table 18.2 Generating and Detecting Reactive Oxygen Species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 821
Product List 18.2 Generating and Detecting Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 823
18.3 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
2,3-Diaminonaphthalene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 825
1,2-Diaminoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826
NBD Methylhydrazine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826
Dichlorodihydrofluorescein Diacetate and Dihydrorhodamine 123 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826
Anti-Nitrotyrosine Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826
S-Nitrosothiol Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826
Griess Reagent Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 827
Measure-iT™ High-Sensitivity Nitrite Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 827
Data Table 18.3 Probes for Nitric Oxide Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 828
Product List 18.3 Probes for Nitric Oxide Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 828
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Chapter 18 — Probes for Reactive Oxygen Species, Including Nitric Oxide Section 18.1 Introduction to Reactive Oxygen Species
18.1 Introduction to Reactive Oxygen Species

Activated oxygen species are produced during a number of physi- Molecular Probes® products include probes that either generate or
ological 1–3 and pathological 4–7 processes. Their effects are through detect various reactive oxygen species (Table 18.1), including singlet
reactions with a large variety of easily oxidizable cellular components, oxygen (1O2), superoxide anion (•O2–), hydroxyl radical (HO•) and
including NADH, NADPH, ascorbic acid, histidine, tryptophan, ty- various peroxides (ROOR’) and hydroperoxides (ROOH). Section 18.2
rosine, cysteine, glutathione, proteins and nucleic acids. 8–12 Reactive describes these probes and their applications in vitro and in vivo.
oxygen species can also oxidize cholesterol and unsaturated fatty ac- The importance of the nitric oxide radical (abbreviated NO) and other
ids, causing membrane lipid peroxidation.7,13,14 Several reviews dis- reactive oxygen species as biological messengers is the focus of intense re-
cuss the chemistry of the different reactive oxygen species and their search.20–22 Section 18.3 is devoted to our probes for promoting, inhibiting
detection.15–19 or detecting nitric oxide production in a variety of experimental systems.
Table 18.1 Reactive oxygen species.

Reactive Oxygen Species Structure Detection Reagents
Hydrogen peroxide H 2O 2 • Carboxy-H2DCFDA (C400) 1–3 • H2DCFDA (D399) 8–11
• CM-H2DCFDA (C6827) 4,5 • Lucigenin (L6868) 12,13
• Dihydrocalcein AM (D23805) • Luminol (L8455) 14
• Dihydrorhodamine 123 (D632, D23806) 6 • RedoxSensor™ Red CC-1 (R14060) 15
• Dihydrorhodamine 6G (D633) 7
Hydroxyl radical * HO• • 3´-(p-Aminophenyl) fluorescein (APF, A36003) • Proxyl fluorescamine (C7924) 17
• 3´-(p-Hydroxyphenyl) fluorescein (HPF, H36004) • TEMPO-9-AC (A7923)
• CM-H2DCFDA (C6827) 16
Hypochlorous acid HOCl • Aminophenyl fluorescein (APF, A36003) • Luminol (L8455) 19–21
• Dihydrorhodamine 123 (D632, D23806) 18
Nitric oxide NO • DAF-FM (D23841) 22,23 • 2,3-Diaminonaphthalene (D7918) 24
• DAF-FM diacetate (D23842, D23844) 22,23 • Luminol (L8455) 25
• DAA (D23840) 24
Peroxyl radical, including both alkylperoxyl ROO• • BODIPY® FL EDA (D2390) 27 • DPPP (D7894) 35–37
and hydroperoxyl 26 radicals (wherein R = H) • BODIPY® 665/676 (B3932) 28 • Luminol (L8455) 38–40
• H2DCFDA (D399) 29–33 • cis-Parinaric acid (P36005) 41,42
• Carboxy-H2DCFDA (C400) 34 • RedoxSensor™ Red CC-1 (R14060) 15
• CM-H2DCFDA (C6827)
Peroxynitrite anion † ONOO– • 3´-(p-Aminophenyl) fluorescein (APF, A36003) • Coelenterazine (C2944) 45
• 3´-(p-Hydroxyphenyl) fluorescein (HPF, H36004) • Dihydrorhodamine 123 (D632, D23806) 43,46–48
• H2DCFDA (D399) 43,44 • Dihydrorhodamine 6G (D633)
• Carboxy-H2DCFDA (C400) • Luminol (L8455) 43,49,50
• CM-H2DCFDA (C6827)
1
Singlet oxygen ‡ O2 • Singlet Oxygen Sensor Green reagent (S36002) • trans-1-(2´-methoxyvinyl)pyrene (M7913) 51,52
Superoxide anion •O2– • Coelenterazine (C2944) 53,54 • MCLA (M23800) 65,66
• Dihydroethidium (D1168, D11347, D23107) 55,56 • MTT (M6494) 67
• Fc OxyBURST® Green assay reagent (F2902) 57,58 • NBT (N6495) 68
• OxyBURST® Green H2DCFDA SE (D2935) 59,60 • RedoxSensor™ Red CC-1 (R14060) 15
• OxyBURST® Green H2HFF BSA (O13291) 61 • TEMPO-9-AC (A7923)
• Lucigenin (L6868) 62,63 • XTT (X6493) 69
• Luminol (L8455) 64
* Hydroxyl radicals can also be photosensitized by malachite green isothiocyanate (M689) or generated by a N-(1,10-phenanthrolin-5-yl)iodoacetamide (P6879) metal–ligand
complex. † 3-Nitrotyrosine, a product of this potent nitrating reagent, can be detected with an anti-nitrotyrosine antibody (A21285). ‡ Singlet oxygen can also be photosensitized by
hypericin (H7476), rose bengal diacetate (R14000) and merocyanine 540 (M24571).
1. Biol Pharm Bull (2000) 23:1153; 2. J Neurosci (1999) 19:9209; 3. J Biol Chem (1996) 271:21505; 4. J Biol Chem (2001) 276:21938; 5. Proc Natl Acad Sci U S A (1997) 94:11557; 6. Biochim Biophys
Acta (1999) 1454:275; 7. Proc Natl Acad Sci U S A (2000) 97:8266; 8. J Biol Chem (2001) 276:514; 9. J Immunol Methods (1989) 117:53; 10. Brain Res (1994) 635:113; 11. J Biol Chem (1999) 274:37111;
12. Analyst (1986) 3:941; 13. J Am Chem Soc (1979) 101:5347; 14. J Bone Miner Res (1992) 7:1139; 15. Free Radic Biol Med (2000) 28:1266; 16. Proc Natl Acad Sci U S A (2001) 98:1643; 17. Anal
Chem (1997) 69:4295; 18. Nitric Oxide (1997) 1:145; 19. Biochim Biophys Acta (1991) 1097:145; 20. Luminescence (1999) 14:239; 21. Am J Physiol (1992) 263:G719; 22. Anal Biochem (2000)
287:203; 23. Angew Chem Int Ed Engl (1999) 38:3209; 24. Neuroreport (1998) 9:4051; 25. Anal Chem (1993) 65:1794; 26. DNA Cell Biol (2002) 21:251; 27. J Biochem Biophys Methods (1997) 35:23;
28. J Agric Food Chem (2000) 48:1150; 29. Toxicol Meth (1994) 4:224; 30. J Biol Chem (2000) 275:40028; 31. Anal Biochem (1983) 134:111; 32. Am J Physiol (1989) 257:C347; 33. Methods Enzymol
(1984) 105:352; 34. J Biol Chem (1998) 273:5294; 35. J Chromatogr (1993) 628:31; 36. Anal Lett (1987) 20:731; 37. Methods Enzymol (1990) 186:157; 38. Free Radic Biol Med (1995) 18:1; 39. Biomed
Chromatogr (1990) 4:131; 40. Lipids (1998) 33:1235; 41. J Biol Chem (1997) 272:12328; 42. Biochem Biophys Res Commun (1998) 244:647; 43. Free Radic Biol Med (2001) 30:463; 44. FEBS Lett
(2000) 468:89; 45. Circ Res (1999) 84:1203; 46. FASEB J 2001; 47. Arch Biochem Biophys (2000) 373:302; 48. FASEB J (2000) 14:1061; 49. J Biol Chem (1996) 271:29223; 50. Arch Biochem Biophys
(1994) 310:352; 51. Biochem Biophys Res Commun (1984) 123:869; 52. Methods Enzymol (1986) 133:569; 53. Anal Biochem (1992) 206:273; 54. Free Radic Biol Med (2000) 29:170; 55. Circ
Res (2001) 88:824; 56. J Biol Chem (2001) 276:17621; 57. J Leukoc Biol (1997) 62:329; 58. J Biol Chem (1995) 270:8328; 59. Immunology (1994) 83:507; 60. J Immunol Methods (1990) 130:223;
61. Biophys J (1998) 75:2577; 62. Free Radic Biol Med (2000) 28:1232; 63. J Biol Chem (1998) 273:2015; 64. J Immunol Methods (1992) 155:151; 65. Free Radic Res (2000) 32:265; 66. Anal Biochem
(1999) 271:53; 67. Free Radic Res Commun (1993) 18:369; 68. Arch Biochem Biophys (1997) 342:275; 69. Plant Physiol (1998) 117:491.
TheMolecular
Probes Handbook:
A Guide Probes
to Fluorescent and
Probes Labeling
and Technologies
™
IMPORTANT NOTICE:described
The products described
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Chapter 18 — Probes for Reactive Oxygen Species, Including Nitric Oxide Section 18.2 Generating and Detecting Reactive Oxygen Species
REFERENCES
1. Chem Res Toxicol (2010) 23:568; 2. Cytometry A (2009) 75:475; 3. J Biol Chem (2006) (2008) 283:15539; 14. Chem Res Toxicol (2008) 21:432; 15. Methods Mol Biol (2010)
281:29011; 4. Nat Clin Pract Cardiovasc Med (2008) 5:811; 5. J Neurosci (2009) 29:9090; 594:57; 16. Methods Cell Biol (2007) 80:355; 17. Free Radic Biol Med (2007) 43:995;
6. Mol Cell (2009) 33:627; 7. Physiol Rev (2004) 84:1381; 8. Mitochondrion (2007) 7:106; 18. J Biochem Biophys Methods (2005) 65:45; 19. Am J Physiol Regul Integr Comp
9. Electrophoresis (2005) 26:2599; 10. Angew Chem Int Ed Engl (2007) 46:561; 11. Br Physiol (2004) 286:R431; 20. Curr Opin Chem Biol (2010) 14:43; 21. Free Radic Biol Med
J Pharmacol (2004) 142:231; 12. Biochem Pharmacol (2003) 66:1527; 13. J Biol Chem (2009) 47:684; 22. Nitric Oxide (2009) 21:92.
18.2 Generating and Detecting Reactive Oxygen Species

2,000
50% D2O
We offer an assortment of Molecular Probes® products for the generation of reactive oxygen
A species (ROS), including singlet oxygen (1O2), superoxide (•O2–), hydroxyl radical (HO•) and
various peroxides (ROORʹ) and hydroperoxides (ROOH) (Table 18.1), as well as for their fluoro-
Fluorescence
metric detection in solution. Although there are no sensors that reversibly monitor the level of
1,000
Tris buffer reactive oxygen species, this section discusses a number of probes that trap or otherwise react
with singlet oxygen, hydroxyl radicals or superoxide. The optical or electron spin properties of
the resulting products can be used as a measure of the presence or quantity of the reactive oxygen
NaN3
species and, in certain cases, can report the kinetics and location of their formation.
0
0 100 200 300
Time (sec)
Generating Singlet Oxygen
300
Singlet oxygen is responsible for much of the physiological damage caused by reactive oxygen
Dihydrorhodamine 123
B species, including nucleic acid modification through selective reaction with deoxyguanosine to
form 8-hydroxydeoxyguanosine 1,2 (8-OHdG). The lifetime of singlet oxygen is sufficiently long
Fluorescence
200
(4.4 microseconds in water 3) to permit significant diffusion in cells and tissues.4 In the labora-
tory, singlet oxygen is usually generated in one of three ways: photochemically from dioxygen
100 (3O2) using a photosensitizing dye (Figure 18.2.1), chemically by thermal decomposition of a
Singlet Oxygen Sensor Green peroxide or dioxetane, or by microwave discharge through an oxygen stream. Singlet oxygen can
0
be directly detected by its characteristic weak chemiluminescence at 1270 nm.4,5
Addition of 50 mU/mL xanthine oxidase
0 100 200 300
Hypericin
Time (sec)
Among the most efficient reagents for generating singlet oxygen is the photosensitizer hyper-
Figure 18.2.1 Fluorescence response and specific- icin (H7476, Figure 18.2.2), a natural pigment isolated from plants of the genus Hypericum. This
ity of Singlet Oxygen Sensor Green reagent (S36002) to heat-stable dye exhibits a quantum yield for singlet oxygen generation in excess of 0.7, as well as
1
O2. A) Fluorescence measurements were made in a spec-
trofluorometer using excitation/emission wavelengths of high photostability, making it an important agent for both anticancer and antiviral research.6,7
488/525 nm for solutions containing: 1 µM Singlet Oxygen
Sensor Green reagent and 10 µM methylene blue in 100 mM
pH 7.5 Tris buffer alone, the singlet oxygen scavenger so-
Rose Bengal Diacetate
dium azide (NaN3, 1 mM), or 50% D2O, which increases the Rose bengal diacetate (R14000) is an efficient, cell-permeant generator of singlet oxygen.8–10
lifetime of 1O2. Measurements were made for 20-second pe- It is an iodinated xanthene derivative that has been chemically modified by the introduction of
riods, with 30-second intervals (indicated by grey bars) be- acetate groups (Figure 18.2.3). These modifications inactivate both its fluorescence and photosen-
tween each measurement. During the 30-second intervals,
the samples were exposed to laser radiation (630–680 nm, sitization properties, while increasing its ability to cross cell membranes. Once inside a live cell,
<5 mW), resulting in methylene blue–photosensitized gen- esterases remove the acetate groups, restoring rose bengal to its native structure. Its intracellular
eration of 1O2. B) Fluorescence measurements were made localization allows rose bengal diacetate to be a very effective photosensitizer.
in a spectrofluorometer using excitation/emission wave-
lengths of 488/525 nm for solutions of 50 mM pH 7 Tris
buffer with 1 mM xanthine containing either 1 µM Singlet Merocyanine 540
Oxygen Sensor Green reagent or dihydrorhodamine 123 Photoirradiation of merocyanine 540 (M24571) produces both singlet oxygen and other
(D632, D23806). After ~20 seconds, 50 mU/mL of xanthine
oxidase (XO) was added. XO catalzyes the oxidation of xan- reactive oxygen species, including oxygen radicals.11–14 Merocyanine 540 is often used as a photo-
thine, producing uric acid and superoxide. Superoxide can sensitizer in photodynamic therapy.15
spontaneously degrade to H2O2.
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Handbook: AA Guide
Guide to
to Fluorescent Probesand
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Technologies
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Appendix onto
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the Appendix onrefer
Detecting Singlet Oxygen

Singlet Oxygen Sensor Green Reagent OH O OH
Unlike other fluorescent and chemiluminescent singlet oxygen detection reagents, the
Singlet Oxygen Sensor Green reagent (S36002) is highly selective for singlet oxygen (1O2); it shows
no appreciable response to other reactive oxygen species, including hydroxyl radical (HO•), su- H3C OH
H3C OH
peroxide (•O2–) and nitric oxide (NO) (Figure 18.2.1). Before reaction with singlet oxygen, this
probe initially exhibits weak blue fluorescence with excitation peaks at 372 and 393 nm and
emission peaks at 395 and 416 nm. In the presence of singlet oxygen, however, it emits a green OH O OH
fluorescence similar to that of fluorescein (excitation/emission maxima ~504/525 nm).
Figure 18.2.2 Hypericin (H7476).
We have observed that the fluorescent product of Singlet Oxygen Sensor Green reagent can
degrade with time in some solutions and that Singlet Oxygen Sensor Green reagent can become
fluorescent at alkaline pH in the absence of singlet oxygen. Nevertheless, with the proper controls
the intensity of the green-fluorescent signal can be correlated with singlet oxygen concentration,
without significant interference from other reactive oxygen species. The Singlet Oxygen Sensor
Green reagent has demonstrated utility for detecting singlet oxygen in solution 16,17 and in plant
tissues.18,19
trans-1-(2´-Methoxyvinyl)pyrene
trans-1-(2´-Methoxyvinyl)pyrene (M7913) can be used to detect picomole quantities of sin-
glet oxygen in chemical and biological systems (Figure 18.2.4), making this compound one of the
most sensitive singlet oxygen probes currently available.20–22 Furthermore, this highly selective Figure 18.2.3 Rose bengal diacetate (R14000).
chemiluminescent probe does not react with other activated oxygen species such as hydroxyl
radical, superoxide or hydrogen peroxide.
O O O
Generating Hydroxyl and Superoxide Radicals

H OCH3 OCH3
C C C C CH
H H H
1
O
2
Hydroxyl and superoxide radicals have been implicated in a number of pathological condi-
tions, including ischemia, reperfusion and aging. The superoxide anion (Table 18.1) may also CL (Em = 465 nm)
play a role in regulating normal vascular function. The hydroxyl radical is a very reactive oxygen Figure 18.2.4 Reaction of trans-1-(2´-methoxyvinyl)pyrene
species 23 that has a lifetime of about 2 nanoseconds in aqueous solution and a radius of diffusion (M7913) with singlet oxygen (1O2), yielding a dioxetane in-
of about 20 Å. Thus, it induces peroxidation only when it is generated in close proximity to its tar- termediate that generates chemiluminescence (CL) upon
decomposition to 1-pyrenecarboxaldehyde.
get. The hydroxyl radical can be derived from superoxide in a Fenton reaction catalyzed by Fe2+
or other transition metals, as well as by the effect of ionizing radiation on dioxygen. Superoxide
is most effectively generated from a hypoxanthine–xanthine oxidase generating system 24–26
(Figure 18.2.1).
(CH 3) 2N N(CH 3) 2 ClO4
Malachite Green C
Malachite green is a nonfluorescent photosensitizer that absorbs at long wavelengths
(~630 nm). Its photosensitizing action can be targeted to particular cellular sites by conjugating
malachite green isothiocyanate (M689, Figure 18.2.5) to specific antibodies.27,28 Enzymes and
N C S
other proteins within ~10 Å of the binding site of the malachite green–labeled antibody can
Figure 18.2.5 Malachite green isothiocyanate (M689).
then be selectively destroyed upon irradiation with long-wavelength light. Studies by Jay and
colleagues have demonstrated that this photoinduced destruction of enzymes in the immediate
vicinity of the chromophore is apparently the result of localized production of hydroxyl radicals,
which have short lifetimes that limit their diffusion from the site of their generation.29
O
1,10-Phenanthroline Iodoacetamide NH C CH2I
Conjugation of the iodoacetamide of 1,10-phenanthroline (P6879, Figure 18.2.6) to thiol-
containing ligands confers the metal-binding properties of this important complexing agent
on the ligand. For example, the covalent copper–phenanthroline complex of oligonucleotides N N
or nucleic acid–binding molecules in combination with hydrogen peroxide acts as a chemical Figure 18.2.6 N-(1,10-phenanthrolin-5-yl)iodoacetamide
nuclease to selectively cleave DNA or RNA.30,31 Hydroxyl radicals or other reactive oxygen spe- (P6879).
cies appear to be involved in this cleavage.32,33
The
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MolecularProbes Handbook:
Probes®
™
A Guide
Handbook: to Fluorescent
A Guide Probes
to Fluorescent and
Probes Labeling
and Technologies
manualinare
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Detecting Hydroxyl
and Superoxide Radicals
MitoSOX™ 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 1–3% 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 mitochondria. 34,35 Increases in cellular su-
Figure 18.2.7 Detection of superoxide in live cells using MitoSOX™ Red superoxide indica- peroxide production have been implicated in cardiovascular diseas-
tor (M36008). Live 3T3 cells were treated with FeTCPP, a superoxide scavenger (right), or left es, including hypertension, atherosclerosis and diabetes-associated
untreated (left). Cells were then labeled with MitoSOX™ Red reagent, which fluoresces when vascular injuries, 36 as well as in neurodegenerative diseases such as
oxidized by superoxide, and nuclei were stained with blue-fluorescent Hoechst 33342. The
mitochondria of untreated cells exhibited red fluorescence, indicating the presence of super- Parkinson disease, Alzheimer disease and amyotrophic lateral scle-
oxide, whereas the mitochondria of treated cells showed minimal fluorescence. rosis (ALS). 35
MitoSOX™ Red mitochondrial superoxide indicator (M36008) is a
OH cationic derivative of dihydroethidum (also known as hydroethidine;
see below) designed for highly selective detection of superoxide in the
H2 N NH2 H2N NH2
O2
mitochondria of live cells (Figure 18.2.7). The cationic triphenylphos-
H N N phonium substituent of MitoSOX™ Red indicator is responsible for
(CH 2) 6 P (CH 2) 6 P
the electrophoretically driven uptake of the probe in actively respir-
3 3
ing mitochondria. Oxidation of MitoSOX™ Red indicator (or dihy-
Figure 18.2.8 Oxidation of MitoSOX™ Red mitochondrial superoxide indicator to 2-hydroxy- droethidium) by superoxide results in hydroxylation at the 2-position
5-(triphenylphosphonium)hexylethidium by superoxide (•O2– ).
(Figure 18.2.8). 2-hydroxyethidium (and the corresponding derivative
of MitoSOX™ Red indicator) exhibits a fluorescence excitation peak at
10,000
~400 nm 37 that is absent in the excitation spectrum of the ethidium
1.6 oxidation product generated by reactive oxygen species other than su-
Absorbance (548 nm)
peroxide. Thus, fluorescence excitation at 400 nm with emission detec-

1.4
Relative fluorescence
1.2
1,000 1.0
0.8
tion at ~590 nm provides optimum discrimination of superoxide from
0.6 other reactive oxygen species 37–39 (Figure 18.2.9).
0.4
100 0.2 Measurements of mitochondrial superoxide generation us-
0
Sodium Nitric Peroxy- ing MitoSOX™ Red indicator in mouse cortical neurons expressing
nitrite oxide nitrite
caspase-cleaved tau microtubule-associated protein have been cor-
10
related with readouts from fluorescent indicators of cytosolic and mi-
tochondrial calcium and mitochondrial membrane potential.40 The
1 relationship of mitochondrial superoxide generation to dopamine
H n
te
n
xi e + ide
xy n
Pe oxy e
e
pe rox ero e
xy n
ng + 2
ad OD
transporter activity, measured using the aminostyryl dye substrate

o D
ad A)
2
Pe oxy P
O
2O
tio
Si itric RP
d
tio
ro ge
de id
Su pe up itrit
ro ge
tri
(n SO
le HR
N DN
le oxi
x
2 O H2
xi ox
di
N +S
di
ni
N H
S n
ro er
2 +
4-Di-1-ASP (D288, Section 12.2), has been investigated in mouse

pe up
t
t
o
Si 2
o
ro id
2O
S
de
ng
H
brain astrocytes.41 MitoSOX™ Red indicator has been used for con-
Su
Su
focal microscopy analysis of reactive oxygen species (ROS) produc-

Figure 18.2.9 Selectivity of the MitoSOX™ Red mitochondrial superoxide indicator tion by mitochondrial NO synthase (mtNOS) in permeabilized cat
(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 µM solu- ventricular myocytes 42 and, in combination with Amplex® Red re-
tion of MitoSOX™ Red reagent and incubated at 37°C for 10 minutes. Excess DNA was add- agent, for measurement of mitochondrial superoxide and hydrogen
ed (unless otherwise noted) and the samples were incubated for an additional 15 minutes peroxide production in rat vascular endothelial cells.43 In addition
at 37°C before fluorescence was measured. The Griess Reagent Kit (G7921) (for nitric oxide,
peroxynitrite, and nitrite standards only; blue bars) and dihydrorhodamine 123 (DHR 123
to imaging and microscope photometry measurements, several flow
(D632); green bars) were employed as positive controls for oxidant generation. Superoxide cytometry applications of MitoSOX™ Red indicator have also been
dismutase (SOD), a superoxide scavenger, was used as a negative control for superoxide. The reported. Detailed protocols for simultaneous measurements of mito-
results show that the MitoSOX™ Red probe (red bars) is readily oxidized by superoxide but
not by the other oxidants.
chondrial superoxide generation and apoptotic markers APC annexin
V (A35110, Section 15.5) and SYTOX® Green (S7020, Section 8.1) in
human coronary artery endothelial cells by flow cytometry have been
H�� H� published by Mukhopadhyay and co-workers.44
H �
CH�CH3
Dihydroethidium (Hydroethidine)
Although dihydroethidium (Figure 18.2.10), which is also called
hydroethidine, is commonly used to analyze respiratory bursts in
Figure 18.2.10 Dihydroethidium (hydroethidine, D1168). phagocytes,45 it has been reported that this probe undergoes significant
TheMolecular
Probes Handbook:
Handbook: AAGuide
Guideto
toFluorescent
™
Fluorescent Probes and Labeling
Probes and LabelingTechnologies
Technologies
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Appendix
referonto
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the Appendix on
oxidation in resting leukocytes, possibly through the uncoupling of mitochondrial oxidative

phosphorylation.46 Cytosolic dihydroethidium exhibits blue fluorescence; however, once this
probe is oxidized to ethidium it intercalates within DNA, staining the cell nucleus a bright fluo-
rescent red 47–49 (Figure 18.2.11). The mechanism of dihydroethidium’s interaction with lyso-
somes and DNA has been described.50 Similar to MitoSOX™ Red mitochondrial superoxide in-
dicator (Figure 18.2.8), dihydroethidium is oxidized by superoxide to 2-hydroxyethidium.37 It is
frequently used for mitochondrial superoxide detection, 51–54 although MitoSOX™ Red indicator
provides more specific mitochondrial localization. Indeed, in some cases researchers have used
dihydroethidium and MitoSOX™ Red indicator to provide discrete indications of cytosolic and
mitochondrial superoxide production, respectively.55
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 Traps Figure 18.2.11 Live bovine pulmonary artery endothelial
Hydroxyl radicals have usually been detected after reaction with spin traps. We offer cells (BPAEC) were incubated with the cell-permeant, weakly
TEMPO-9-AC (A7923, Figure 18.2.12) and proxyl fluorescamine 56–59 (C7924, Figure 18.2.13), blue-fluorescent dihydroethidium (D1168, D11347, D23107)
and the green-fluorescent mitochondrial stain, MitoTracker®
two fluorogenic probes for detecting hydroxyl radicals 60 and superoxide. Each of these molecules Green FM® (M7514). Upon oxidation, red-fluorescent ethid-
contains a nitroxide moiety that effectively quenches its fluorescence. However, once TEMPO- ium accumulated in the nucleus.
9-AC or proxyl fluorescamine traps a hydroxyl radical or superoxide, its fluorescence is restored
and the radical’s electron spin resonance signal is destroyed, making these probes useful for
detecting radicals either by fluorescence or by electron spin resonance spectroscopy. TEMPO-9- N
AC has been reported to detect glutathionyl radicals but not phenoxyl radicals.61 Proxyl fluores-
camine can be used to detect the methyl radicals that are formed by reacting hydroxyl radicals
with DMSO.59 Radical-specific scavengers (Table 18.2)—such as the superoxide-specific p-ben- C O
zoquinone and superoxide dismutase 62 or the hydroxyl radical–specific mannitol and dimethyl- NH
sulfoxide (DMSO) 56,63,64—can be used to identify the detected species.
H3C CH3
Chemiluminescent and Chromogenic Reagents for Detecting Superoxide H3 C N CH3
In the absence of apoaequorin, the luminophore coelenterazine (C2944) produces chemi- O
luminescence in response to superoxide generation in cells, organelles, bacteria 65 and tissues.66
Figure 18.2.12 4-((9-acridinecarbonyl)amino)-2,2,6,6-tet-
Unlike luminol, coelenterazine exhibits luminescence that does not depend on the activity of ramethylpiperidin-1-oxyl, free radical (TEMPO-9-AC, A7923).
cell-derived myeloperoxidase and is not inhibited by azide.67
In addition to coelenterazine, we offer MCLA (M23800, Figure 18.2.14) for detecting su-
peroxide.68 MCLA and coelenterazine are superior alternatives to lucigenin 65 (L6868) for this O
application because lucigenin can reportedly sensitize superoxide production, leading to false- O C O K
positive results.69–73 An additional advantage of MCLA is that its pH optimum for luminescence OH
generation is closer to the physiological near-neutral range than are the pH optima of luminol
and lucigenin.74 N
CH2
H3C CH3
Table 18.2 Scavengers of reactive oxygen species (ROS). H3C N CH3

O
ROS Scavenger (Working Concentration) References
Hydrogen peroxide (H2O2) Sodium pyruvate (10 mM), DMTU* (10 mM) 1 Figure 18.2.13 5-(2-carboxyphenyl)-5-hydroxy-1-((2,2,5,5-
Hydroxyl radical (HO•) Mannitol (20–100 mM), DMSO (0.28 M) † 1 tetramethyl-1-oxypyrrolidin-3-yl)methyl)-3-phenyl-2-pyrro-
lin-4-one, potassium salt (proxyl fluorescamine, C7924).
Nitric oxide (NO) Carboxy-PTIO (C7912; 100 µM) 1
Peroxyl radical (ROO•) Trolox ‡ (10–100 µM), α-tocopherol (10–100 µM) 2,3
Peroxynitrite anion (ONOO–) Ebselen § (10–100 µM), uric acid (100 µM) 1,4 O
Singlet oxygen (1O2) Sodium azide (1–10 mM) 1 CH3 HC�
Superoxide anion (•O2–) MnTBAP ** (100 µM), Tiron †† (10 mM) 5,6 �
CH3O �
* DMTU = N,Nʹ-dimethylthiourea. Disproportionation by catalase is also widely used for suppression of H2O2. † 0.28
M DMSO = 2% (v/v). The reactivity of HO• is so high that it can be argued that the actions of these reagents must be �
indirect. ‡ Trolox = 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. § Ebselen = 2-phenyl-1,2-benzisoselenazol- H
3(2H)-one. ** MnTBAP = manganese(III)-tetrakis(4-benzoic acid)porphyrin. †† Tiron = 4,5-dihydroxybenzene-1,3-disulfonate.
1. J Biol Chem (2007) 282:30452; 2. Nitric Oxide (2006) 15:163; 3. Biochim Biophys Acta (2004) 1636:136; 4. J Biol Chem (2004) Figure 18.2.14 2-methyl-6-(4-methoxyphenyl)-3,7-dihydro-
279:4425; 5. Circ Res (2004) 94:37; 6. Bioorg Med Chem (2002) 10:3013. imidazo1,2-apyrazin-3-one, hydrochloride (MCLA, M23800).
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Nitro blue tetrazolium salt (NBT, N6495; Table 18.3) and other free radical–mediated damage in cells. To directly assess the extent
tetrazolium salts are chromogenic probes useful for superoxide deter- of lipid peroxidation, researchers either measure the amount of lipid
mination.75,76 The superoxide sensitivity of tetrazolium salts can be a hydroperoxides directly or detect the presence of secondary reaction
confounding factor in their more common applications for cell viability products 91–93 (e.g., 4-hydroxy-2-nonenal or malonaldehyde; see below).
and proliferation assays.77 Peroxyl radicals are formed by the decomposition of various
peroxides and hydroperoxides, including lipid hydroperoxides. The
hydroperoxyl radical is also the protonated form of superoxide, and
Detecting Peroxides, Peroxyl approximately 0.3% of the superoxide in the cytosol is present as this
Radicals and Lipid Peroxidation protonated radical.94 Experimentally, peroxyl radicals, including alkyl-
peroxyl (ROO•) and hydroperoxyl (HOO•) radicals, are generated from
In peroxisomes, H2O2 is produced by several enzymes that use mo- compounds such as 2,2´-azobis(2-amidinopropane) and from hydro-
lecular oxygen to oxidize organic compounds. This H2O2 is then used peroxides such as cumene hydroperoxide.
by catalase to oxidize other substrates, including phenols, formic acid,
formaldehyde and alcohol. In liver and kidney cells, these oxidation cis-Parinaric Acid
reactions are important for detoxifying a variety of compounds in the Fluorescence quenching of the fatty acid analog cis-parinaric acid
bloodstream.78–81 However, H2O2 also plays a role in neurodegenerative (P36005) has been used in several lipid peroxidation assays,95–97 in-
and other disorders through induction of apoptosis 82 and DNA strand cluding quantitative determinations in live cells.98,99 Parinaric acid’s
breaks,83 modification of intracellular Ca 2+ levels and mitochondrial extensive unsaturation (Figure 18.2.15) makes it quite susceptible to
potential, and oxidation of glutathione. In addition, H2O2 is released oxidation if not rigorously protected from air.100 Consequently, we of-
from cells during hypoxia.84 fer cis-parinaric acid in a 10 mL unit size of a 3 mM solution in de-
Peroxidation of unsaturated lipids affects cell membrane proper- oxygenated ethanol (P36005); if stored protected from light under an
ties,85 signal transduction pathways,86,87 apoptosis and the deteriora- inert argon atmosphere at –20°C, this stock solution should be stable
tion of foods and other biological compounds.88 Lipid hydroperoxides for at least 6 months. During experiments, we advise handling parinaric
have been reported to accumulate in oxidatively stressed individuals, acid samples under inert gas and preparing solutions using degassed
including HIV-infected patients.89 Lipid peroxidation may also be re- buffers and solvents. Parinaric acid is also somewhat photolabile and
sponsible for aging, as well as for pathological processes such as drug- undergoes photodimerization when exposed to intense illumination,
induced phototoxicity and atherosclerosis,90 and is often the cause of resulting in loss of fluorescence.101
Diphenyl-1-Pyrenylphosphine
H H Hydroperoxides in lipids, serum, tissues and foodstuffs can be di-
C C H O rectly detected using the fluorogenic reagent diphenyl-1-pyrenylphos-
CH3CH2 C C H
H C C (CH 2) 7 C OH phine 102,103 (DPPP, D7894). DPPP is essentially nonfluorescent until
H C C oxidized to a phosphine oxide by peroxides; in vitro, DPPP remains
H H
nonfluorescent in the presence of hydroxyl radicals generated by the
Figure 18.2.15 cis-parinaric acid (P36005).
Cu2+-ascorbate method.104 DPPP has previously been used to detect
Table 18.3 Tetrazolium salts for detecting redox potential in living cells and tissues.
Color of Water Solubility
Cat. No. Tetrazolium Salt Formazan of Formazan Applications
M6494 (MTT) 3-(4,5-Dimethylthiazol-2-yl)-2,5- purple no • Superoxide generation by fumarate reductase 1 and nitric oxide synthase 2
diphenyltetrazolium bromide • Mitochondrial dehydrogenase activity 3
• Cell viability and proliferation 4–9
• Neuronal cell death 10
• Platelet activation 11
• Tumor cell adhesion 12 and invasion 13
• Multidrug resistance 14
• In vitro toxicity testing 15–17
N6495 (NBT) Nitro blue tetrazolium chloride deep blue no • Superoxide generation by xanthine oxidase 18
• Neutrophil oxidative metabolism 19,20
• NADPH diaphorase activity 21–23
• Succinic dehydrogenase histochemistry 24
X6493 (XTT) 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)- orange yes • Antifungal susceptibility 25
2H-tetrazolium-5-carboxanilide • Drug sensitivity of cells 26
• Parasitic nematode viability 27
• Tumor cell cytotoxicity 28
1. J Biol Chem (1995) 270:19767; 2. J Biol Chem (1994) 269:12589; 3. Cytometry (1992) 13:532; 4. Biotechniques (1998) 25:622, 626; 5. J Immunol Methods (1994) 168:253; 6. Anal Biochem
(1993) 214:190; 7. J Immunol Methods (1993) 164:149; 8. Anal Biochem (1992) 205:8; 9. J Immunol Methods (1986) 89:271; 10. J Cell Biol (1995) 128:201; 11. J Immunol Methods (1993) 159:253;
12. J Immunol Methods (1993) 164:255; 13. Cancer Res (1994) 54:3620; 14. Leuk Res (1992) 16:1165; 15. Biosci Biotechnol Biochem (1992) 56:1472; 16. J Immunol Methods (1991) 144:141;
17. J Immunol Methods (1990) 131:165; 18. J Reprod Fertil (1993) 97:441; 19. Clin Chim Acta (1993) 221:197; 20. J Leukoc Biol (1993) 53:404; 21. Neurosci Lett (1993) 155:61; 22. Proc Natl Acad Sci
U S A (1991) 88:7797; 23. Proc Natl Acad Sci U S A (1991) 88:2811; 24. Histochemistry (1982) 76:381; 25. Antimicrob Agents Chemother (1992) 36:1619; 26. Cancer Res (1988) 48:4827; 27. Parasitology
(1993) 107:175; 28. J Immunol Methods (1992) 147:153.
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Handbook: AAGuide
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™
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Appendix onto
picomole levels of hydroperoxides by HPLC.105,106 Its solubility in lipids horseradish peroxidase (HRP) to investigate reoxygenation injury in rat
makes DPPP quite useful for detecting hydroperoxides in the mem- hepatocytes.134,135 In these experiments, it is thought that the primary
branes of live cells 104,107,108 and in low-density lipoprotein particles.109 species being detected is hydrogen peroxide. In addition, luminol has
been employed to detect peroxynitrite generated from the reaction of
BODIPY® 581/591 C: A Ratiometric nitric oxide and superoxide.136–138
Lipid Peroxidation Sensor
The BODIPY® 581/591 C11 fatty acid (D3861, Figure 18.2.16) is a Detecting 4-Hydroxy-2-Nonenal
sensitive fluorescent reporter for lipid peroxidation, undergoing a shift Formation of 4-hydroxy-2-nonenal (HNE) from linoleic acid is a
from red to green fluorescence emission upon oxidation of the phen- major cause of lipid peroxidation–induced toxicity. Several reagents
ylbutadiene segment of the fluorophore.110 This oxidation-dependent for the direct fluorometric detection of aldehydes are described in
emission shift enables fluorescence ratio imaging of lipid peroxidation Section 3.3. The biotinylated hydroxylamine ARP (A10550, Section
in live cells.111–113 Other common applications of BODIPY® 581/591 4.2) is particularly useful for this purpose.139 Biotinylation using click
C11 include fluorometric assays of antioxidant efficacy in plasma 114,115 chemistry coupling (Section 3.1) enables affinity purification of HNE-
and in lipid vesicles.116 The oxidation and nitroxidation products of modified proteins.140
this BODIPY® fatty acid have been characterized by mass spectrom-
etry.117,118 Based on mass spectrometry analysis of oxidation products,
MacDonald and co-workers report that BODIPY® 581/591 C11 is more Detecting Peroxides and Peroxidases
sensitive to oxidation than endogenous lipids, and therefore tends to with Amplex® Red Reagents
overestimate oxidative damage and underestimate antioxidant protec-
tion effects.119 Amplex® Red Reagent: Stable Substrate
Peroxyl radicals have also been detected in erythrocyte and red for Peroxidase Detection
blood cell membranes using BODIPY® FL EDA 120 (D2390, Section In the presence of horseradish peroxidase (HRP), Amplex® Red
3.4), a water-soluble BODIPY® dye, or BODIPY® FL hexadecanoic acid reagent (10-acetyl-3,7-dihydroxyphenoxazine, A12222, A22177; Figure
(D3821, Section 13.2). BODIPY® FL hexadecanoic acid exhibits the red 18.2.17) reacts with H2O2 in a 1:1 stoichiometry to produce highly
shift common to the fluorescence of lipophilic BODIPY® dyes when they fluorescent resorufin 141 (R363, Section 10.1, Figure 18.2.18). Amplex®
are concentrated, permitting ratiometric measurements of hydroxyl Red reagent has greater stability, yields less background and produces
radical production and allowing the onset of lipid peroxidation in live a red-fluorescent product that is more readily detected than the similar
cells to be monitored.121 reduced methylene blue derivatives commonly used for colorimetric
determination of lipid peroxides in plasma, sera, cell extracts and a va-
Other Scavengers for Peroxyl Radicals riety of membrane systems.142–144
The fluorescence of several other probes is lost following interac-
tion with peroxyl radicals. Lipophilic fluorescein dyes such as hexa-
decanoylaminofluorescein 122 (H110, Section 13.5) and fluorescein-
labeled phosphatidylethanolamine (F362, Section 13.2) have been HO O OH
useful for detecting peroxyl radical formation in membranes and in
solution. Phycobiliproteins, such as B-phycoerythrin, R-phycoerythrin N
and allophycocyanin (P800, P801, A803, A819; Section 6.4), and pheno- C CH3
lic dyes such as fluorescein (F1300, F36915; Section 10.1) are extensively O
used as substrates in total antioxidant capacity assays of plasma and Figure 18.2.17 Amplex® Red reagent (A12222).
foods.115,123,124
Luminol
Although luminol (L8455) is not useful for detecting superoxide
in live cells,125 it is commonly employed to detect peroxidase- or metal Amplex® Red Resorufin
Peroxidase
ion–mediated oxidative events.126–128 Used alone, luminol can detect ox- HO O OH HO O O
idative events in cells rich in peroxidases, including granulocytes 129–132 N N

and spermatozoa.133 This probe has also been used in conjunction with C CH
3
O
H O O
2 2 2
CH OH CH OH
2 2
OH
O O
OH O OH
HO Glucose Oxidase HO
OH OH
Figure 18.2.18 Principle of coupled enzymatic assays using Amplex® Red reagent. Oxidation
of glucose by glucose oxidase results in generation of H2O2, which is coupled to conversion
Figure 18.2.16 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3- of the Amplex® Red reagent to fluorescent resorufin by HRP. The detection scheme shown
undecanoic acid (BODIPY® 581/591 C11, D3861). here is used in the Amplex® Red Glucose/Glucose Oxidase Assay Kit (A22189).
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Amplex® Red reagent has been used to detect the release of H2O2 from activated human
20,000
500
leukocytes,141,145 to measure the activity of monoamine oxidase in bovine brain tissue,146 to
400 Amplex®
UltraRed
demonstrate the extracellular production of H2O2 produced by UV light stimulation of human
15,000
300
200
keratinocytes 147–149 and for microplate assays of H2O2 and lipid hydroperoxide generation by
isolated mitochondria.51,68,150 Amplex® Red reagent is available in a single 5 mg vial (A12222)
Fluorescence
100
10,000
0
0 10 20 30 or packaged as a set of 10 vials, each containing 10 mg of the substrate, for high-throughput
Amplex®
Red
screening applications (A22177).
5,000
Amplex® UltraRed Reagent: Brighter and More
Sensitive than the Amplex® Red Reagent
0
0 200 400 600 800 1,000 Amplex® UltraRed reagent (A36006) improves upon the performance of Amplex® Red re-
Hydrogen peroxide (nM) agent, offering brighter fluorescence and enhanced sensitivity on a per-mole basis in horseradish
Figure 18.2.19 Detection of H2O2 using Amplex® UltraRed
peroxidase or horseradish peroxidase–coupled enzyme assays (Figure 18.2.19). Fluorescence of
reagent (red squares) or Amplex® Red reagent (blue tri- oxidized Amplex® UltraRed reagent is also less sensitive to pH (Figure 18.2.20), and the substrate
angles). Reactions containing 50 µM Amplex® UltraRed and its oxidation product exhibit greater stability than Amplex® Red reagent in the presence of
or Amplex® Red reagent, 1 U/mL HRP and the indicated
amount of H2O2 in 50 mM sodium phosphate buffer, pH 7.4,
H2O2 or thiols such as dithiothreitol (DTT). Like Amplex® Red reagent, nonfluorescent Amplex®
were incubated for 30 minutes at room temperature. The UltraRed reagent reacts with H2O2 in a 1:1 stoichiometric ratio to produce a brightly fluorescent
inset shows the sensitivity and linearity of the Amplex® and strongly absorbing reaction product (excitation/emission maxima ~568/581 nm) (Figure
UltraRed assay at low levels of H2O2.
18.2.21). Although the primary applications of the Amplex® UltraRed reagent are enzyme-linked
immunosorbent assays (ELISAs; see Zen™ Myeloperoxidase ELISA Kit below) and in vitro anti-
oxidant capacity assays,151 it is also frequently used (in combination with HRP) to detect H2O2
production by isolated mitochondria 152 and cell cultures.153,154
Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit

The Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188) provides a simple,
Fluorescence emission
Amplex® UltraRed
sensitive, one-step assay for detecting H 2O2 or the activity of horseradish peroxidase either
Amplex® Red by measuring fluorescence with a fluorescence-based microplate reader or a fluorometer
(Figure 18.2.22) or by measuring absorption with an absorption-based microplate reader or
a spectrophotometer. The Amplex® Red peroxidase substrate can detect the presence of ac-
tive peroxidases and the release of H 2O2 from biological samples, including cells and cell
extracts. 51,141,155,156
The Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit contains:
1 2 3 4 5 6 7 8 9 10
pH
• Amplex® Red reagent
• Dimethylsulfoxide (DMSO)
Figure 18.2.20 Comparison of pH-dependent fluorescence
of the products derived from oxidation of Amplex® UltraRed • Horseradish (HRP)
reagent (solid blue circles) and Amplex® Red reagent (open • H2O2 for use as a positive control
blue squares). Fluorescence intensities were measured using • Concentrated reaction buffer
excitation/emission of ~570/585 nm.
• Detailed protocols
Each kit provides sufficient reagents for approximately 500 assays using a fluorescence- or
absorption-based microplate reader and a reaction volume of 100 µL per assay. Several additional
kits that utilize the Amplex® Red peroxidase substrate to detect H2O2 in coupled enzymatic reac-
tions are described in Section 10.5.
Amplex® Red Xanthine/Xanthine Oxidase Assay Kit

Xanthine oxidase (E.C. 1.2.3.2) plays a key role in the production of free radicals, in-
Absorption
cluding superoxide, in the body. The Amplex® Red Xanthine/Xanthine Oxidase Assay Kit
(A22182) provides an ultrasensitive method for detecting xanthine or hypoxanthine or for
monitoring xanthine oxidase activity. In the assay, xanthine oxidase catalyzes the oxidation
of purine nucleotides, hypoxanthine or xanthine, to uric acid and superoxide. In the reac-
tion mixture, the superoxide spontaneously degrades to H 2O2 , which in the presence of HRP
reacts stoichiometrically with Amplex® Red reagent to generate the red-fluorescent oxida-
400 450 500 550 600 650 700
tion product, resorufin. Resorufin has absorption and fluorescence emission maxima of ap-
Wavelength (nm)
proximately 571 nm and 585 nm (Figure 18.2.23), respectively, and because the extinction
Figure 18.2.21 Absorption and fluorescence emission spectra
of the product generated by horseradish peroxidase–mediated
coefficient is high (54,000 cm–1M–1), the assay can be performed either fluorometrically or
oxidation of the Amplex® UltraRed reagent in pH 7.5 buffer. spectrophotometrically.
The
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Handbook: AAGuide
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™
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and Labeling
Technologies
812 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
The Amplex® Red Xanthine/Xanthine Oxidase Assay Kit (A22182) contains:
• Amplex® Red reagent • Xanthine oxidase from buttermilk

• Dimethylsulfoxide (DMSO) • Hypoxanthine
• Horseradish peroxidase (HRP) • Xanthine
• H 2 O2 • Detailed protocols
• Concentrated reaction buffer
Each kit provides sufficient reagents for approximately 400 assays using either a fluores-
cence- or absorption-based microplate reader and a reaction volume of 100 µL per assay.
In healthy individuals, xanthine oxidase is present in appreciable amounts only in the
liver and jejunum. In various liver disorders, however, the enzyme is released into circula-
tion. Therefore, determination of serum xanthine oxidase levels serves as a sensitive indicator of Figure 18.2.23 Absorption and fluorescence emission
acute liver damage such as jaundice. The Amplex® Red xanthine/xanthine oxidase assay has been spectra of resorufin in pH 9.0 buffer.
used as a marker of recovery from exercise stress.157 Previously, researchers have utilized chemi-
luminescence or absorbance to monitor xanthine oxidase activity. The Amplex® Red Xanthine/
Xanthine Oxidase Assay Kit permits the detection of xanthine oxidase in a purified system at
levels as low as 0.1 mU/mL by fluorescence (Figure 18.2.24). This kit can also be used to detect
as little as 200 nM hypoxanthine or xanthine (Figure 18.2.25), and, when coupled to the purine
nucleotide phosphorylase enzyme, to detect inorganic phosphate.158
EnzChek® Myeloperoxidase (MPO) Activity Assay Kit

Myeloperoxidase (MPO, EC 1.11.1.7) 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 13.5 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 inflammatory diseases, autoimmune diseases and can- Figure 18.2.24 Detection of xanthine oxidase using
cer. MPO is also experimentally and clinically important for distinguishing myeloid from lym- the Amplex® Red Xanthine/Xanthine Oxidase Assay Kit
phoid leukemia and, due to its role in the pathology of atherogenesis, has been advocated as a (A22182). Each reaction contained 50 µM Amplex® Red
reagent, 0.2 U/mL horseradish peroxidase, 0.1 mM hypo-
prognostic marker of cardiovascular disease. xanthine and the indicated amount of xanthine oxidase in
The ferric, or native, MPO reacts with hydrogen H 2O2 to form the active inter mediate 1X reaction buffer. After 30 minutes, fluorescence was mea-
MPO-I, which oxidizes chloride (Cl–) to HOCl; these reactions make up the chlorination cycle sured in a fluorescence microplate reader using excitation
at 530 ± 12.5 nm and detection at 590 ± 17.5 nm. A back-
(Figure 18.2.26). MPO also oxidizes a variety of substrates, including phenols and anilines, ground of 65 fluorescence units was subtracted from each
via the classic peroxidation cycle. The relative concentrations of chloride and the reducing data point. The inset shows the assay’s sensitivity and linear-
substrate determine whether MPO uses hydrogen peroxide for chlorination or peroxida- ity at low hypoxanthine concentrations.
tion. Assays based on measurement of chlorination activity are more specific for MPO than
those based on peroxidase substrates such as tetramethylbenzidine (TMB).
3,500
3,000
2,500
Fluorescence
2,000 500
400
1,500
300
1,000 200
100 Figure 18.2.25 Detection of hypoxanthine using the

500 0 Amplex® Red Xanthine/Xanthine Oxidase Assay Kit
0 0.1 0.2 0.3 0.4
(A22182). Each reaction contained 50 µM Amplex® Red
0
0 1 2 3 4 5 6 7 reagent, 0.2 U/mL horseradish peroxidase, 20 mU/mL xan-
thine oxidase and the indicated amount of hypoxanthine in
HRP (mU/mL)
1X reaction buffer. Reactions were incubated at 37°C. After
Figure 18.2.22 Detection of HRP using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188). Reactions con- 30 minutes, fluorescence was measured in a fluorescence mi-
taining 50 µM Amplex® Red reagent, 1 mM H2O2 and the indicated amount of HRP in 50 mM sodium phosphate buffer, pH 7.4, croplate reader using excitation at 530 ± 12.5 nm and detec-
were incubated for 30 minutes at room temperature. Fluorescence was measured with a fluorescence microplate reader using tion at 590 ± 17.5 nm. A background of 54 fluorescence units
excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. Background fluorescence (3 units), determined for a was subtracted from each data point. The inset shows the as-
no-HRP control reaction, was subtracted from each value. The inset shows the sensitivity of the assay at very low levels of HRP. say’s sensitivity and linearity at low enzyme concentrations.
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™
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or moreUse
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The EnzChek® Myeloperoxidase (MPO) Activity Assay Kit (E33856) pro-

3′-(p-aminophenyl) fluorescein (APF) fluorescein vides assays for rapid and sensitive determination of both chlorination and
peroxidation activities of MPO in solution and in cell lysates 159–161 (Figure
HOCl Cl– 18.2.26). For detection of chlorination, the kit provides nonfluorescent
3´-(p-aminophenyl) fluorescein (APF), which is selectively cleaved by hypo-
chlorite (–OCl) to yield fluorescein. Peroxidation is detected using nonfluo-
rescent Amplex® UltraRed reagent, which is oxidized by the H2O2-generated
redox intermediates MPO-I and MPO-II to form a fluorescent product. The
EnzChek® Myeloperoxidase Activity Assay Kit can be used to continuously
Chlorination activity
MPO MPO-I detect these activities at room temperature over a broad dynamic range (1.5
Peroxidation activity H2O2 to 200 ng/mL) (Figure 18.2.27). The speed (30 minutes), sensitivity and mix-
AHr AH2 and-read convenience make this kit ideal for measuring MPO activities and
for high-throughput screening for MPO-specific inhibitors.
MPO-II Each EnzChek® Myeloperoxidase (MPO) Activity Assay Kit contains:
AH2 AHr
• 3´-(p-aminophenyl) fluorescein (APF)
• Amplex® UltraRed reagent
• Human myeloperoxidase (MPO) standard
AHr + AHr A + AH2
• Chlorination inhibitor
Figure 18.2.26 Schematic diagram for detection of chlorination and peroxidation activity • Peroxidation inhibitor
of MPO using the EnzChek® Myeloperoxidase (MPO) Activity Assay Kit (E33856). AH2 rep-
resents the nonfluorescent Amplex® UltraRed substrate, and A represents its fluorescent
• Hydrogen peroxide (H2O2)
oxidation product. • Phosphate-buffered saline (PBS)
Sufficient reagents are provided to perform 200 assays for chlorination

and 200 assays for peroxidation activity in a 96-well fluorescence microplate
format (100 µL per assay).
A 7,000
Zen™ Myeloperoxidase (MPO) ELISA Kit
The Zen™ Myeloperoxidase (MPO) ELISA Kit (Z33857) provides a com-
5,600
prehensive set of components for accurate and sensitive quantitation of hu-

4,200
man MPO in a variety of biological samples, including human serum. This
2,800 200
sandwich immunoassay utilizes the Amplex® UltraRed reagent, a fluoro-
genic substrate for horseradish peroxidase (HRP) that reacts with H2O2 in a
100
1,400 1:1 stoichiometric ratio to produce the brightly fluorescent and strongly ab-
0
0 3 6 9 12 sorbing Amplex® UltraRed oxidation product (excitation/ emission maxima
0
0 40 80 120 160 200 ~568/581 nm). Because the Amplex® UltraRed product has long-wavelength
MPO (ng/mL) emission, there is little interference from the blue or green autofluorescence
4,000 found in most biological samples. With a high extinction coefficient, good
B
quantum efficiency and resistance to autooxidation, the fluorescence-based
3,200
Amplex® UltraRed reagent delivers better sensitivity and a broader assay range
2,400 than colorimetric reagents.
Each Zen™ Myeloperoxidase (MPO) ELISA Kit contains:
1,600 200
100 • Amplex® UltraRed reagent

800
0
• Concentrated phosphate-buffered saline (PBS)
0 3 6 9 12
0
0 40 80 120 160 200
• Horseradish peroxidase (HRP) labeled goat anti–rabbit IgG antibody
MPO (ng/mL)
• Amplex® stop reagent
Figure 18.2.27 Typical standard curves for detection of MPO using the APF-based • Hydrogen peroxide (H2O2)
chlorination assay (A) and Amplex® UltraRed–based peroxidation assay (B) provided • MPO standard
in the EnzChek® Myeloperoxidase (MPO) Activity Assay Kit (E33856). Reactions were • Bovine serum albumin (BSA)
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 • Tween® 20
was measured with a fluorescence microplate reader using fluorescence excitation • Mouse anti-MPO antibody (capture antibody)
and emission at 485 and 530 nm, respectively, for the APF assay, or excitation and • Rabbit anti-MPO antibody (detection antibody)
emission at 530 and 590 nm, respectively, for the Amplex® UltraRed assay. The back-
ground fluorescence measured for each zero-MPO control reaction was subtracted • Zen™ microplates for oriented capture anitbody coating
from each fluorescence measurement before plotting. • Detailed protocols
The
Handbook: A
A Guide
Guide to
to Fluorescent
™
Probesand
Technologies
IMPORTANT NOTICE: The products described in this manual aremanual
coveredare
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by Limited Use Label License(s). Please refer to thePlease
Appendix on to
814 IMPORTANT NOTICE : The products described in this covered one or more Limited Use Label License(s).
Sufficient reagents are provided for 200 assays in a microplate format, using a 100 µL per well
reaction volume. The Zen™ Myeloperoxidase (MPO) ELISA Kit can be used to detect from 0.2 to 3,600
100 ng/mL MPO at room temperature (Figure 18.2.28).
2,400
Assaying Oxidative Activity in Live Cells 1,000
The generation of reactive oxygen species (ROS) is inevitable for aerobic organisms and, 1,200 500
in healthy cells, occurs at a controlled rate. Under conditions of oxidative stress, however, ROS 0
0 0.5 1 1.5 2 2.5 3 3.5
production is dramatically increased, resulting in subsequent alteration of membrane lipids,
0
proteins and nucleic acids. Oxidative damage of these biomolecules is associated with aging and 0 20 40 60 80 100
with a variety of pathological events, including atherosclerosis, carcinogenesis, ischemic reperfu- MPO (ng/mL)
sion injury and neuorodegenerative disorders.40,162,163 Figure 18.2.28 Typical standard curve for detection of
MPO using the Zen™ Myeloperoxidase (MPO) ELISA Kit
Assaying oxidative activity in live cells with fluorogenic, chemiluminescent or chromogenic (Z33857). The sandwich ELISA was carried out as described
probes is complicated by the frequent presence of multiple reactive oxygen species in the same in the protocol using a mouse anti-MPO primary capture an-
cell. Scavengers and enzymes such as superoxide dismutase and catalase are useful knockdown tibody, MPO standards ranging from 0.2 ng/mL to 100 ng/mL,
and a rabbit anti-MPO detection antibody.
reagents for triaging the optical response of ROS probes (Table 18.2). Quantitative analysis can
be further hindered due to: 1) the high intracellular concentration of glutathione, which can form
thiyl or sulfinyl radicals or otherwise trap or reduce oxygen species; 164 2) the variable concentra-
tion of metals, which can either catalyze or inhibit radical reactions; and 3) the presence of other
free radical–quenching agents such as spermine.165
Fluorescein, rhodamine and various other dyes can be chemically reduced to colorless,
nonfluorescent leuco dyes. These "dihydro" derivatives are readily oxidized back to the parent
dye by reactive oxygen species and thus can serve as fluorogenic probes for detecting oxidative
activity in cells and tissues.166–168 Oxidation also occurs spontaneously, albeit slowly, in air and
via photosensitization when illuminated for fluorescence excitation.169,170 Careful storage and
handling, as well as minimizing the duration and intensity of light exposure, are particularly
recommended when using these dyes. In general, dihydrofluorescein and dihydrorhodamine do Figure 18.2.29 2’,7’-dichlorodihydrofluorescein diacetate
not discriminate between the various reactive oxygen species. It has been reported that dichlo- (2’,7’-dichlorofluorescin diacetate; H2DCFDA, D399).
rodihydrofluorescein (H2DCF) and dihydrorhodamine 123 react with intracellular hydrogen
peroxide in a reaction mediated by peroxidase, cytochrome c or Fe2+,23,171 and these leuco dyes
also serve as fluorogenic substrates for peroxidase enzymes (Section 10.5).
Dichlorodihydrofluorescein Diacetate
The cell-permeant 2´,7´-dichlorodihydrofluorescein diacetate (H2DCFDA, D399; Figure
18.2.29), also known as dichlorofluorescin diacetate, is commonly used to detect the generation
of reactive oxygen intermediates in neutrophils and macrophages.172–176 Upon cleavage of the ac-
etate groups by intracellular esterases and subsequent oxidation, the nonfluorescent H2DCFDA
is converted to the highly fluorescent 2´,7´-dichlorofluorescein (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 radicals.170 In a cell-free system, H2DCF has been
shown to be oxidized to DCF by peroxynitrite anion (ONOO –), by horseradish peroxidase (in the
absence of H2O2) and by Fe2+ (in the absence of H2O2).177 Furthermore, the oxidation of H2DCF
by Fe2+ in the presence of H2O2 was reduced by the HO• radical scavenger formate and the iron
chelator deferoxamine.177 In addition, DCF itself can act as a photosensitizer for H2DCFDA
oxidation, both priming and accelerating the formation of DCF.170 Because the oxidation of
DCF and H2DCFDA appears to also generate free radicals, their use for measuring free radical
production must be carefully controlled.178
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 8.1) with H2DCFDA to simultaneously monitor oxidant production and
cell injury.179 H2DCFDA has been used to visualize oxidative changes in carbon tetrachloride–
perfused rat liver 180 and in venular endothelium during neutrophil activation,181 as well as to
examine the effect of ischemia and reperfusion in lung and heart tissue.182,183 Using H2DCFDA,
researchers characterized hypoxia-dependent peroxide production in Saccharomyces cerevisiae
as a possible model for ischemic tissue destruction.184 In neutrophils, H2DCFDA has proven
TheMolecular
The MolecularProbes
Probes® Handbook:
A Guide Probes
to Fluorescent and
Probes Labeling
and Technologies
™
manualin are
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manual are
bycovered by oneLimited
or moreUse
Limited UseLicense(s).
Label License(s).
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IMPORTANT NOTICE : The products in this one or more Label to the Appendix
O O useful for flow cytometric analysis of nitric oxide, forming a product that has spectral properties
CH3CO O OCCH3 identical to those produced when it reacts with hydrogen peroxide.185 In this study, H2DCFDA’s
reaction with nitric oxide was blocked by adding the nitric oxide synthase inhibitor NG-methyl-
Cl Cl L-arginine (L-NMMA) to the cell suspension.185 2´,7´-Dichlorofluorescein—the oxidation prod-
H
COCH2OCCH3 uct of H2DCF—can reportedly be further oxidized to a phenoxyl radical in a horseradish peroxi-
O O dase–catalyzed reaction, and this reaction may complicate the interpretation of results obtained
CH3COCH2OC with this probe in cells undergoing oxidative stress.186 Although other more specialized ROS
O O probes have been—and continue to be—developed, H2DCFDA and its chloromethyl derivative
Figure 18.2.30 6-carboxy-2’,7’-dichlorodihydrofluorescein CM-H2DCFDA remain the most versatile indicators of cellular oxidative stress.187
diacetate, di(acetoxymethyl ester), C2938.
Improved Versions of H2DCFDA
Intracellular oxidation of H2DCF tends to be accompanied by leakage of the product, 2´,7´-di-
chlorofluorescein,188 which may make quantitation or detection of slow oxidation difficult. To en-
hance retention of the fluorescent product, we offer the carboxylated H2DCFDA analog 189 (carboxy-
H2DCFDA, C400), which has two negative charges at physiological pH, and its di(acetoxymethyl
ester) analog, 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) 190
(C2938, Figure 18.2.30, Figure 18.2.31). Upon cleavage of the acetate and ester groups by intracel-
lular esterases and oxidation, both analogs form carboxydichlorofluorescein (C368, Section 14.3),
with additional negative charges that impede its leakage out of the cell.
The fluorinated analog 5-(and 6-)carboxy-2´,7´-difluorodihydrofluorescein diacetate (car-
boxy-H2DFFDA, C13293) is also useful for visualizing oxidative bursts and inflammatory and
infectious processes.191 As the oxidation potential of deacetylated carboxy-H2DFFDA is more
positive than that of the corresponding chloro compound carboxy-H2DCFDA, its oxidant sen-
sitivity profile is presumably shifted; however, it is not known if this difference is large enough to
have practical utility. The diacetate derivatives of the dichloro- and difluorodihydrofluoresceins
Figure 18.2.31 Bovine pulmonary artery endothelial
(BPAEC) cells were initially stained with the reactive oxygen
are quite stable. When used for intracellular applications, the acetates are cleaved by endogenous
species (ROS) indicator, 6-carboxy-2’,7’-dichlorodihydro- esterases, releasing the corresponding dichloro- or difluorodihydrofluorescein derivative. If,
fluorescein diacetate, di(acetoxymethyl ester) (C2938). After however, these nonfluorescent diacetate derivatives are used for in vitro assays, they must first
a 30-minute incubation, the cells were washed and then
incubated simultaneously with FM® 5-95 (T23360) and
be hydrolyzed with mild base to form the colorless probe.150
Hoechst 33342 (H1399, H3570, H21492) in phosphate-buff- In addition, we have developed 5-(and 6-)chloromethyl-2´,7´-dichlorodihydrofluorescein
ered saline (PBS) for an additional 5 minutes before washing diacetate, acetyl ester (CM-H2DCFDA, C6827; Figure 18.2.32; Figure 18.2.33), which is a chloro-
and mounting in PBS. The red-fluorescent FM® 5-95 appears
to stain both the plasma membrane and early endosomes;
methyl derivative of H2DCFDA that exhibits much better retention in live cells.192,193 As with our
the green-fluorescent, oxidized carboxydichlorofluores- other chloromethyl derivatives (see the description of our CellTracker™ probes in Section 14.2),
cein localizes to the cytoplasm; and the blue-fluorescent CM-H2DCFDA passively diffuses into cells, where its acetate groups are cleaved by intracellular
Hoechst 33342 dye stains the nucleus.
esterases and its thiol-reactive chloromethyl group reacts with intracellular glutathione and other
thiols. Subsequent oxidation yields a fluorescent adduct that is trapped inside the cell, thus facili-
tating long-term studies.192,193 Among its many applications, CM-H2DCFDA has been used to:
O O
CH3CO O OCCH3 • Analyze FOXO3 transcriptional control of oxidative stress 194,195
• Assess ROS-mediated cytotoxicity and apoptosis 196–199
Cl
H
Cl
• Detect hydroxyl radicals associated with estrogen-induced DNA damage 200
COCCH3
• Monitor time courses of ROS generation in neurons and brain slices 192,201
O O
6 • Analyze ROS production in chromosomally unstable human–hamster hybrid cells using
ClH2C flow cytometry 202
5
Figure 18.2.32 5-(and-6)-chloromethyl-2’,7’-dichlorodihydro-

fluorescein diacetate, acetyl ester (CM-H2DCFDA, C6827). 120
80
Figure 18.2.33 An oxidative burst was detected by flow cytometry
Counts
of cells labeled with 5-(and 6-)chloromethyl-2’,7’-dichlorodihydroflu-

orescein diacetate, acetyl ester (CM-H2DCFDA, C6827). Jurkat cells
were incubated with 100 nM CM-H2DCFDA. The cells were washed
40
and resuspended in either phosphate-buffered saline (PBS, red) or
PBS with 0.03% H2O2 (blue). The samples were analyzed on a flow
cytometer equipped with a 488 nm argon-ion laser and a 525 ±
10 nm bandpass emission filter.
0
100 101 102 103 104
Green fluorescence
The
TheMolecular
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
Guideto
toFluorescent
™
Fluorescent Probes
Probes and
and Labeling
Technologies
816 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
Image-iT® LIVE Green Reactive Oxygen Species Detection Kit

The Image-iT® LIVE Green Reactive Oxygen Species Detection Kit (I36007) provides the
key reagents for detecting reactive oxygen species (ROS) in live cells (Figure 18.2.34), including:
• Carboxy-H2DCFDA (5-(and 6-)carboxy-2´,7´-dichlorodihydrofluorescein diacetate)

• Hoechst 33342
• tert-butyl hydroperoxide (TBHP)
• Detailed protocols for fluorescence microscopy assays
This assay is based on carboxy-H2DCFDA (5-(and 6-)carboxy-2´,7´-dichlorodihydrofluorescein

diacetate), a reliable fluorogenic marker for reactive oxygen species in live cells.203,204 In addition to
carboxy-H2DCFDA, this kit provides the common inducer of ROS production, tert-butyl hydro-
peroxide (TBHP), as a positive control 205–208 and the blue-fluorescent, cell-permeant nucleic acid
stain Hoechst 33342. Oxidatively stressed and nonstressed cells can be effectively distinguished by
fluorescence microscopy using this combination of dyes and the protocol provided.209–211
Aminophenyl Fluorescein and Hydroxyphenyl Fluorescein

Developed by Nagano, 3´-(p-aminophenyl) fluorescein (APF, A36003) and 3´-(p-hydroxy-
phenyl) fluorescein (HPF, H36004) provide greater selectivity and stability than dichlorodihy-
drofluorescein diacetate (H2DCFDA, D399) for ROS detection.212 H2DCFDA is probably the
most commonly used reagent for detecting intracellular reactive oxygen species despite its lack of
specificity and tendency to spontaneously photooxidize. The nonfluorescent H2DCFDA becomes
fluorescent in the presence of a wide variety of reactive oxygen species including, but not lim- Figure 18.2.34 Detection of oxidative stress in live cells
using the Image-iT® LIVE Green Reactive Oxygen Species
ited to, peroxyl (ROO•) and hydroxyl (HO•) radicals and the peroxynitrite anion (ONOO –). In (ROS) Detection Kit (I36007). Live bovine pulmonary artery
contrast, APF and HPF show much more limited reactivity and greater resistance to light-in- endothelial cells were treated with tert-butyl hydroperoxide
duced oxidation (Table 18.4). Both of these fluorescein derivatives are essentially nonfluores- to induce oxidative stress (bottom) or were left untreated
(top). Cells were then labeled with carboxy-H2DCFDA, which
cent until they react with the hydroxyl radical,60 peroxynitrite anion or singlet oxygen 16 (Figure fluoresces when oxidized by ROS, and nuclei were stained
18.2.35). APF will also react with the hypochlorite anion (–OCl), making it possible to use APF with blue-fluorescent Hoechst 33342. The stressed cells
exhibited green fluorescence, signaling an increase in ROS,
whereas the untreated cells showed minimal fluorescence.
X=O 3ʹ-(p-hydroxyphenyl) fluorescein (HPF)
XH
X = NH 3ʹ-(p-aminophenyl) fluorescein (APF)
O O O O O O
ROS
COO COO
X O
Nonfluorescent Fluorescent
Figure 18.2.35 Detection of reactive oxygen species (ROS) with 3’-(p-hydroxyphenyl) fluorescein (HPF, H36004) and
3’-(p-aminophenyl) fluorescein (APF, A36003).
Table 18.4 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 µM H2O2 <1 2 190
Hydroxyl radical (HO•) 100 µM ferrous perchlorate (II) and 1 mM of H2O2 1200 730 7400
Hypochlorite anion (–OCl) 3 µM (final) –OCl 3600 6 86
Nitric oxide (NO) 100 µM 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-methyl-1-triazene (NOC-7) <1 6 150
Peroxyl radical (ROO•) 100 µM 2,2’-azobis(2-amidinopropane), dihydrochloride (AAPH) 2 17 710
Peroxynitrite anion (ONOO–) 3 µM (final) ONOO 560 120 6600
Singlet oxygen (1O2) 100 µM 3-(1,4-dihydro-1,4-epidioxy-1-naphthyl)propionic acid 9 5 26
Superoxide anion (•O2–) 100 µM KO2 6 8 67
Autooxidation 2.5 hours exposure to fluorescent light source <1 <1 2000
* 10 µm of APF, HPF or DCF (2’,7’-dichlorofluorescein) were added to sodium phosphate buffer (0.1 M, pH 7.4); ROS were generated as indicated; and fluorescence was measured using
excitation/emission wavelengths of 490/515 nm (for APF and HPF) or 500/520 nm (for DCF). DCF was obtained by hydrolysis of H2DCFDA with base as described in J Biol Chem (2003) 278:3170;
dihydrofluorescein diacetates are colorless and nonfluorescent until both of the acetate groups are hydrolyzed and the products are subsequently oxidized to fluorescein derivatives.
TheMolecular
The MolecularProbes
Probes® Handbook:
A Guide Probes
to Fluorescent and
Probes Labeling
and Technologies
™
manualin are
this covered
manual are
or moreUse
Label License(s).
PleasePlease
on on
and HPF together to selectively detect the hypochlorite anion (Section 21.2). In the presence
O O O O
�CH3COCH�OCCH�� CH�COCH�OCCH3��
of these specific reactive oxygen species, both APF and HPF yield a bright green-fluorescent
O CH� CH� O product (excitation/emission maxima ~490/515 nm) and are compatible with all fluorescence
CH3CO O OCCH3 instrumentation capable of visualizing fluorescein. Using APF, researchers have been able to
detect the hypochlorite anion generated by activated neutrophils, a feat that has not been possible
H with traditional ROS indicators.212
COCH�OCCH3
O O
Dihydrocalcein AM
Figure 18.2.36 Dihydrocalcein, AM (D23805). We have combined the superior retention of calcein (the intracellular product of calcein
AM hydrolysis in viable cells) and the oxidation sensitivity of the dihydrofluoresceins to yield
the probe dihydrocalcein AM (D23805, Figure 18.2.36), provided specially packaged as a set of
20 vials, each containing 50 µg. The oxidant sensitivity profile of dihydrocalcein AM has been
O O
characterized relative to that of H2DCFDA 213 and of alkaline elution assays of oxidative DNA
CH3CO O OCCH3
modification.214
O
C� C�
H
C O �
OxyBURST® Green Reagents
Fc OxyBURST® Green assay reagent (F2902) was developed in collaboration with Elizabeth
O
O Simons of Boston University to monitor the oxidative burst in phagocytic cells using fluorescence
instrumentation. The Fc OxyBURST® Green assay reagent comprises bovine serum albumin
Figure 18.2.37 2’,7’-dichlorodihydrofluorescein diacetate,
succinimidyl ester (OxyBURST® Green H2DCFDA, SE, D2935). (BSA) that has been covalently linked to dichlorodihydrofluorescein (H2DCF) and then com-
plexed with purified rabbit polyclonal anti-BSA antibodies. When these immune complexes bind
to Fc receptors, the nonfluorescent H2DCF molecules are internalized within the phagovacuole
and subsequently oxidized to green-fluorescent dichlorofluorescein (DCF); see Section 16.1 for
a more complete description.
OxyBURST® Green H2HFF BSA (O13291) is a sensitive fluorogenic reagent for de-
tecting extracellular release of oxidative products in a spectrofluorometer or a fluores-
cence microscope. This reagent comprises BSA that has been covalently linked to dihydro-
2´,4,5,6,7,7´-hexafluorofluorescein (H2HFF), a reduced dye with improved stability. Unlike
Fc OxyBURST® Green assay reagent, OxyBURST® Green H2HFF BSA is not complexed with
Figure 18.2.38 Dihydrorhodamine 123 (D632). IgG. OxyBURST® Green H2HFF BSA provides up to 1000-fold greater sensitivity than conven-
tional methods based on spectrophotometric detection of superoxide dismutase–inhibitable re-
duction of cytochrome c.215,216
Amine-Reactive OxyBURST® Green Reagent

As an alternative to Fc OxyBURST® Green assay reagent and OxyBURST® Green H2HFF BSA,
we offer the amine-reactive OxyBURST® Green H2DCFDA succinimidyl ester (2´,7´-dichloro-
dihydrofluorescein diacetate, SE; D2935; Figure 18.2.37), 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 microspheres.217,218
Following conjugation to amines, the two acetates of OxyBURST® Green H2DCFDA can be
removed by treatment with hydroxylamine at neutral pH to yield the dihydrofluorescein conju-
gate. OxyBURST® Green H2DCFDA conjugates are nonfluorescent until they are oxidized to the
corresponding fluorescein derivatives.
Dihydrorhodamine 123
Figure 18.2.39 Live bovine pulmonary artery endothe- Dihydrorhodamine 123 (D632, D23806; Figure 18.2.38, Figure 18.2.39) is the uncharged
lial cells (BPAEC) were first stained with LysoTracker® Red
DND-99 (L7528). Then, a solution of dihydrorhodamine 123 and nonfluorescent reduction product of the mitochondrion-selective dye rhodamine 123 (R302,
(D632, D23806) and Hoechst 33258 (H1398, H3569, H21491) R22420; Section 12.2). This leuco dye passively diffuses across most cell membranes where it is
was added and allowed to incubate with the cells for an oxidized to cationic rhodamine 123 (Figure 18.2.40), which localizes in the mitochondria. Like
additional 10 minutes before the cells were subsequently
washed and visualized. The green-fluorescent oxidation H2DCF, dihydrorhodamine 123 does not directly detect superoxide, 219 but rather reacts with
product (rhodamine 123, R302) localized primarily to the hydrogen peroxide in the presence of peroxidase, cytochrome c or Fe2+.23 However, dihydrorho-
mitochondria. The red-fluorescent LysoTracker® Red DND- damine 123 also reacts with peroxynitrite, the anion formed when nitric oxide reacts with super-
99 stain accumulated in the lysosomes, and the blue-flu-
orescent Hoechst 33258 dye stained the nuclei. The image oxide.171 Peroxynitrite, which may play a role in many pathological conditions, has been shown
was acquired with filters appropriate for DAPI, fluorescein to react with sulfhydryl groups, DNA and membrane phospholipids, as well as with tyrosine and
and the Texas Red® dye. The image was deconvolved us- other phenolic compounds.220–223
ing Huygens software (Scientific Volume Imaging, www.svi.
nl). 3D reconstruction was performed using Imaris software
Dihydrorhodamine 123 has been used to investigate reactive oxygen intermediates pro-
(Bitplane AG, www.bitplane.com). duced by human and murine phagocytes,45 activated rat mast cells 224 and vascular endothelial
The
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™
Probesand
Technologies
coveredare
by one or more
Appendix on to
tissues.225,226 It has also been employed to study the role of the CD14 cell-surface marker in H2O2 H�� O �H� C�
production by human monocytes.227
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 C OCH3
DMSO solution is recommended when only small quantities are to be used at a time. O
A Longer-Wavelength Reduced Rhodamine Figure 18.2.40 Rhodamine 123 (R302).
Intracellular oxidation of dihydrorhodamine 6G (D633) yields rhodamine 6G (R634), which

localizes in the mitochondria of live cells (Section 12.2). As compared with rhodamine 123, this
cationic oxidation product has longer-wavelength spectra, making it especially useful in multi- (CH 3) 2N O N(CH 3) 2
color applications and in autofluorescent cells and tissues. Dihydrorhodamine 6G has been used
for fluorescence microplate assays of granulocyte activation 228 and for analysis of ROS levels in H
human umbilical vein endothelial (HUVEC) cells by flow cytometry.194
Reduced MitoTracker® Probes

Two of our MitoTracker® probes—MitoTracker® Orange CM-H2TMRos (M7511, Figure CH2Cl
18.2.41) and MitoTracker® Red CM-H2XRos (M7513, Figure 18.2.42)—are chemically reactive Figure 18.2.41 MitoTracker® Orange CM-H2TMRos (M7511).
reduced rosamines. Unlike MitoTracker® Orange CMTMRos and MitoTracker® Red CMXRos
(M7510, M7512; Section 12.2), the reduced versions of these probes do not fluoresce until they
enter an actively respiring cell, where they are oxidized by reactive oxygen species to the fluo-
rescent mitochondrion-selective probe and then sequestered in the mitochondria. Although
� O �
CM-H2TMRos and CM-H2XRos are widely used as indicators of mitochondrial reactive oxygen
species,229–231 their fluorescence cannot be unambiguously associated with the site of oxidant
generation, as the cationic charge that drives their electrophoretic sequestration in active mito- H
chondria is only present after the probe has been oxidized. This same caveat also applies to di-
hydrorhodamine 123 and dihydrorhodamine 6G. Probes such as MitoSOX™ Red mitochondrial
superoxide indicator resolve this ambiguity by having their oxidant response and mitochondrial
CH�C�
localization functions associated with different structural elements (Figure 18.2.8).
Figure 18.2.42 MitoTracker® Red CM-H2XRos (M7513).
RedoxSensor™ Red CC-1 Stain
RedoxSensor™ Red CC-1 stain (2,3,4,5,6-pentafluorotetramethyldihydrorosamine, R14060;
Figure 18.2.43) passively enters live cells and is subsequently oxidized in the cytosol to a red- �CH3�� O ��CH3��
fluorescent product (excitation/emission maxima ~540/600 nm), which then accumulates in the
mitochondria. Alternatively, this nonfluorescent probe may be transported to the lysosomes
H
where it is oxidized. The differential distribution of the oxidized product between mitochondria � �
and lysosomes appears to depend on the redox potential of the cytosol.232–234 In proliferating
cells, mitochondrial staining predominates; whereas in contact-inhibited cells, the staining is � �
primarily lysosomal (Figure 18.2.44). �
Figure 18.2.43 RedoxSensor™ Red CC-1 (R14060).

Glutathiolation Detection with BioGEE
Biotinylated glutathione ethyl ester (BioGEE, G36000; Figure 18.2.45) 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 identification of oxidation-sensitive proteins.235,236 Once these cells are fixed and
H H
� O
�
H
�H
�CH�� H
O C O O O
Figure 18.2.44 Cellular proliferation state determines the
�HCHCH�CH� C �HCH C �HCH� C OCH�CH3 distribution of the oxidized product of RedoxSensor™ Red
C O CH��H CC-1 (R14060). Normal rat kidney (NRK) cells in different
growth states were stained with RedoxSensor™ Red CC-1. In
OH proliferating cells (left panel), the oxidized dye accumulates
in mitochondria. In quiescent cells (right panel), the oxi-
Figure 18.2.45 Glutathione ethyl ester, biotin amide (BioGEE, G36000). dized product localizes in the lysosomes.
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permeabilized, glutathiolation levels can be detected with a fluorescent streptavidin conjugate

(Section 7.6, Table 7.9) using either flow cytometry or fluorescence microscopy. Proteins glutathi-
olated with BioGEE can be captured using streptavidin agarose (S951, Section 7.6) and analyzed
� � � CH3 by mass spectrometry or by western blotting methods.198,237
� ��
� � CH3 Tetrazolium Salts: Chromogenic Redox Indicators
Figure 18.2.46 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
Tetrazolium salts—especially MTT (M6494, Figure 18.2.46)—are widely used for detecting
tetrazolium bromide), M6494. the redox potential of cells for viability, proliferation and cytotoxicity assays. Upon reduction,
these water-soluble colorless compounds form uncharged, brightly colored formazans. Several
of the formazans precipitate out of solution and are useful for histochemical localization of the
site of reduction or, after solubilization in organic solvent, for quantitation by standard spectro-
photometric techniques. The 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 18.3. Our Vybrant® MTT
Cell Proliferation Assay Kit (V13154, Section 15.4) provides a means of counting metabolically
active cells; this Vybrant® MTT assay can detect from 2000 to 250,000 cells, depending on the
cell type and conditions. See also Section 15.2 for additional cell applications of tetrazolium salts.
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276:35253; 63. Proc Natl Acad Sci U S A (1990) 87:1620; 64. Eur J Biochem (1994) 144. Biochem Int (1985) 10:205; 145. J Immunol Methods (1997) 202:133; 146. Anal
221:695; 65. Anal Biochem (2004) 324:45; 66. Am J Physiol Heart Circ Physiol Biochem (1997) 253:169; 147. J Invest Dermatol (1999) 112:751; 148. Free Radic Biol
(2009) 296:H840; 67. Anal Biochem (1992) 206:273; 68. J Biol Chem (2009) 284:46; Med (1999) 27:1197; 149. J Invest Dermatol (1998) 110:966; 150. J Biol Chem (2006)
69. Arch Biochem Biophys (2000) 373:447; 70. Circ Res (1999) 84:1203; 71. Biochem 281:39766; 151. J Pharmacol Exp Ther (2008) 324:970; 152. Methods Enzymol
Biophys Res Commun (1998) 248:382; 72. J Biol Chem (1998) 273:33972; (2009) 456:381; 153. Am J Physiol Lung Cell Mol Physiol (2007) 292:L1289;
73. Inflammation (1996) 20:151; 74. J Biolumin Chemilumin (1997) 12:277; 75. Anal 154. J Biol Chem (2007) 282:14186; 155. J Immunol (2010) 184:582;
Biochem (2001) 298:337; 76. J Biol Chem (1998) 273:6041; 77. Anal Biochem (2003) 156. J Neurochem (2001) 79:266; 157. Am J Physiol Endocrinol Metab (2002)
313:338; 78. Biochim Biophys Acta (1975) 385:232; 79. Biochem Biophys Res 282:E474; 158. Anal Biochem (2003) 320:292; 159. Exp Gerontol (2008) 43:563;
Commun (1989) 163:836; 80. Biochim Biophys Acta (1989) 981:235; 81. FEBS Lett 160. Eur J Immunol (2007) 37:467; 161. Blood (2007) 109:4716; 162. J Neurosci
(1984) 169:169; 82. J Cell Biol (1998) 273:26900; 83. Neurochem Res (1997) 22:333; (2009) 29:9090; 163. J Neurosci (2007) 27:1129; 164. Nat Protoc (2009) 4:1790;
The
TheMolecular
MolecularProbes®
Probes Handbook:
Handbook: AAGuide
Guideto
toFluorescent
Technologies
™

in thisare covered bycovered
Appendix
referonto
820 IMPORTANT NOTICE : The products described manual are by one or more Limited Use Label License(s).
the Appendix on
REFERENCES—continued
165. Proc Natl Acad Sci U S A (1992) 89:11426; 166. Arch Toxicol (1994) 68:582; 202. Cancer Res (2003) 63:3107; 203. Am J Physiol Heart Circ Physiol (2000)
167. Brain Res (1994) 635:113; 168. Chem Res Toxicol (1992) 5:227; 169. Biochem 279:H2424; 204. J Natl Cancer Inst (1999) 91:1138; 205. Lipids (2001) 36:57;
Biophys Res Commun (2003) 304:619; 170. Free Radic Biol Med (2002) 33:938; 206. Cancer Res (2001) 61:1392; 207. Histochem Cell Biol (2003) 120:319; 208. Am
171. Methods Enzymol (2008) 441:261; 172. J Immunol Methods (1993) 159:173; J Physiol (1997) 272:C1286; 209. Nature (2007) 447:686; 210. J Neurochem (2006)
173. J Immunol Methods (1993) 159:131; 174. Exp Cell Res (1993) 209:375; 98:1474; 211. J Biol Chem (2006) 281:6760; 212. J Biol Chem (2003) 278:3170;
175. Cytometry (1992) 13:615; 176. Cytometry (1992) 13:525; 177. Biochem 213. Free Radic Res (2004) 38:1257; 214. Toxicol In Vitro (2007) 21:1552; 215. J Biol
Pharmacol (2003) 65:1575; 178. Free Radic Biol Med (2006) 40:968; 179. Methods Chem (1980) 255:1874; 216. J Clin Invest (1978) 61:1081; 217. J Cell Physiol (1993)
Enzymol (1994) 233:128; 180. Lab Invest (1991) 64:167; 181. Am J Physiol (1993) 156:428; 218. J Immunol Methods (1990) 130:223; 219. Eur J Biochem (1993)
264:H881; 182. Lab Invest (1994) 70:579; 183. Free Radic Res Commun (1992) 217:973; 220. Free Radic Res (2000) 33:771; 221. Methods Mol Biol (1998) 100:215;
16:217; 184. Cytometry (1993) 14:287; 185. J Leukoc Biol (1992) 51:496; 186. J Biol 222. Mol Med (2000) 6:779; 223. Methods Enzymol (1994) 233:229; 224. APMIS
Chem (1999) 274:28161; 187. Methods Mol Biol (2010) 594:57; 188. Free Radic Biol (1994) 102:474; 225. Atherosclerosis (2003) 169:19; 226. Circ Res (2004) 94:239;
Med (1994) 16:509; 189. Free Radic Biol Med (2007) 43:300; 190. Oncogene (2009) 227. J Immunol Methods (2006) 316:27; 228. Nat Med (2009) 15:300; 229. Nat Clin
28:2690; 191. Cancer Res (2009) 69:5860; 192. J Neurosci (2001) 21:1949; Pract Cardiovasc Med (2008) 5:811; 230. Biochemistry (2006) 45:7237; 231. Diabetes
193. J Neurosci (2007) 27:11315; 194. J Biol Chem (2009) 284:14476; 195. J Biol (2006) 55:120; 232. Am J Pathol (2009) 174:101; 233. Am J Physiol Renal Physiol
Chem (2008) 283:25692; 196. Chem Res Toxicol (2010) 23:568; 197. Toxicol Sci (2007) 292:F523; 234. Free Radic Biol Med (2000) 28:1266; 235. Amino Acids (2007)
(2008) 103:335; 198. Nat Immunol (2008) 9:866; 199. Toxicol In Vitro (2008) 33:51; 236. Biochemistry (2000) 39:11121; 237. J Biol Chem (2009) 284:22213.
22:1392; 200. J Biol Chem (2009) 284:8633; 201. Pflugers Arch (2009) 458:937;
DATA TABLE 18.2 GENERATING AND DETECTING REACTIVE OXYGEN SPECIES

Cat. No. MW Storage Soluble Abs EC Em Solvent Notes
A7923 376.48 F,D,L DMSO 358 11,000 424 MeOH 1
A12222 257.25 FF,D,A DMSO 280 6000 none pH 8 2
A22177 257.25 FF,D,A DMSO 280 6000 none pH 8
A36003 423.42 RO,L DMF 454 24,000 515 pH 9 3, 4
A36006 ~300 FF,D,A DMSO 293 11,000 none pH 8 5
B3932 448.32 F,L DMSO, CHCl3 665 161,000 676 MeOH
C400 531.30 F,D DMSO, EtOH 290 5600 none MeCN 6
C2938 675.43 F,D,AA DMSO 291 5700 none MeOH 6
C2944 423.47 FF,D,LL,AA MeOH 429 7500 see Notes pH 7 7, 8, 9
C6827 577.80 F,D,AA DMSO 287 9100 none MeOH 6
C7924 487.62 F,D,L DMSO, H2O 385 5800 485 pH 7 1
C13293 498.39 F,D DMSO, EtOH 290 5500 none MeCN 10
D399 487.29 F,D DMSO, EtOH 258 11,000 none MeOH 6
D632 346.38 F,D,L,AA DMF, DMSO 289 7100 none MeOH 11, 12
D633 444.57 F,D,L,AA DMF, DMSO 296 11,000 none MeOH 11, 12
D1168 315.42 FF,L,AA DMF, DMSO 355 14,000 see Notes MeCN 11, 13
D2935 584.37 F,D,AA DMF 258 11,000 none MeOH 6
D3861 504.43 F,L DMSO 582 140,000 591 MeOH 14
D7894 386.43 F,D,LL MeCN 358 29,000 none MeOH 15
D11347 315.42 FF,L,AA DMF, DMSO 355 14,000 see Notes MeCN 11, 13
D23107 315.42 FF,D,L,AA DMSO 355 14,000 see Notes MeCN 13, 16
D23805 1068.95 F,D DMSO 285 5800 none MeCN 17
D23806 346.38 F,D,L,AA DMSO 289 7100 none MeOH 12, 16
F2902 see Notes RR,L,AA H2O <300 none 3, 18, 19
G36000 561.67 F,D DMSO <300 none
H7476 504.45 F,D,L DMSO, DMF 591 37,000 594 EtOH
H36004 424.41 RO,L DMF 454 28,000 515 pH 9 3, 4
L6868 510.50 L H2O 455 7400 505 H2O 20, 21
L8455 177.16 D,L DMF 355 7500 411 MeOH 21
M689 485.98 F,DD,L DMF, DMSO 629 75,000 none MeCN 22
M6494 414.32 D,L H2O, DMSO 375 8300 none MeOH 23, 24
M7511 392.93 F,D,L,AA DMSO 235 57,000 none MeOH 11, 12
M7513 497.08 F,D,L,AA DMSO 245 45,000 none MeOH 11, 12
M7913 258.32 F,L DMF, DMSO 352 30,000 401 MeOH 25
M23800 291.74 FF,D,LL,AA DMSO 430 8400 546 MeOH 26
M24571 569.67 D,L DMSO, EtOH 555 143,000 578 MeOH
M36008 759.71 FF,L,AA DMSO 356 10,000 410 MeCN 11, 27
N6495 817.65 D,L H2O, DMSO 256 64,000 none MeOH 23
O13291 ~66,000 F,D,L,AA H2O <300 none 28
P800 ~240,000 RR,L see Notes 546 2,410,000 575 pH 7 29
P801 ~240,000 RR,L see Notes 565 1,960,000 578 pH 7 29
continued on next page
TheMolecular
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A Guide Probes
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Probes Labeling
and Technologies
DATA TABLE 18.2 GENERATING AND DETECTING REACTIVE OXYGEN SPECIES—continued

P6879 363.16 F,D,L DMSO 270 28,000 none CHCl3 30
P36005 276.42 FF,LL,AA EtOH 304 77,000 416 MeOH 3, 31
R14000 1057.75 F,D DMSO 313 9700 none MeOH 32
R14060 434.41 F,D,L,AA DMSO 239 52,000 none MeOH 11, 33
S36002 ~600 F,D,L DMSO 508 105,000 528 pH 7 34, 35
X6493 674.53 F,D H2O, DMSO 286 15,000 none MeOH 36
For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.
Notes
1. Fluorescence of A7923 and C7924 is weak. Reaction of the nitroxide moiety with superoxide or hydroxyl radicals results in increased fluorescence without a spectral shift. (Anal Biochem (1993)
212:85)
2. Peroxidase-catalyzed reaction of the Amplex® Red reagent (A12222, A22177) with H2O2 produces fluorescent resorufin (R363). Resorufin is unstable in the presence of thiols such as dithiothreitol
(DTT) and 2-mercaptoethanol. (Bioorg Chem (1998) 26:63)
3. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."
4. Fluorescence of A36003 and H36004 is extremely weak. Highly fluorescent fluorescein F1300 is generated upon oxidation. (J Biol Chem (2003) 278:3170)
5. Peroxidase-catalyzed reaction of the Amplex® UltraRed reagent (A36006) with H2O2 yields a fluorescent product with Abs = 568 nm (EC = 57,000 cm–1M–1), Em = 581 nm in pH 7.5 buffer.
6. Dihydrofluorescein diacetates are colorless and nonfluorescent until both of the acetate groups are hydrolyzed and the products are subsequently oxidized to fluorescein derivatives. The materi-
als contain less than 0.1% of oxidized derivative when initially prepared. The oxidation products of C400, C2938, C6827, D399 and D2935 are 2´,7´-dichlorofluorescein derivatives with spectra
similar to C368 (Section 14.3).
7. C2944 emits chemiluminescence (Em = 466 nm) on oxidation by superoxide. (Anal Biochem (1992) 206:273)
8. Do NOT dissolve in DMSO.
9. Aqueous solutions of coelenterazine (>1 mM) can be prepared in pH 7 buffer containing 50 mM 2-hydroxypropyl-β-cyclodextrin. (Biosci Biotechnol Biochem (1997) 61:1219)
10. Difluorodihydrofluorescein diacetates are colorless and nonfluorescent. Acetate hydrolysis and subsequent oxidation generate a fluorescent 2´,7´-difluorofluorescein derivative with spectra
similar to O6146 (Section 14.3).
11. This compound is susceptible to oxidation, especially in solution. Store solutions under argon or nitrogen. Oxidation may be induced by illumination.
12. These compounds are essentially colorless and nonfluorescent until oxidized. Oxidation products (in parentheses) are as follows: D632 and D23806 (R302); D633 (R634); M7511 (M7510); M7513
(M7512).
13. Dihydroethidium has blue fluorescence (Em ~420 nm) until oxidized to ethidium (Em ~605 nm). The reduced dye does not bind to nucleic acids. (FEBS Lett (1972) 26:169)
14. Oxidation of the polyunsaturated butadienyl portion of the BODIPY® 581/591 dye results in a shift of the fluorescence emission peak from ~590 nm to ~510 nm. (Methods Enzymol (2000)
319:603, FEBS Lett (1999) 453:278)
15. Oxidation of D7894 occurs rapidly in solution when illuminated. The oxidation product is strongly fluorescent. Em = 379 nm.
16. This product is supplied as a ready-made solution in DMSO with sodium borohydride added to inhibit oxidation.
17. D23805 is colorless and nonfluorescent until the AM ester groups are hydrolyzed and the resulting leuco dye is subsequently oxidized. The final product is calcein (C481).
18. F2902 is essentially colorless and nonfluorescent until oxidized. A small amount (~5%) of oxidized material is normal and acceptable for the product as supplied. The oxidation product is
fluorescent (Abs = 495 nm, Em = 524 nm). (J Immunol Methods (1990) 130:223)
19. This product consists of a dye–bovine serum albumin conjugate (MW ~66,000) complexed with IgG in a ratio of approximately 1:4 mol:mol (BSA:IgG)
20. L6868 has much stronger absorption at shorter wavelengths (Abs = 368 nm (EC = 36,000 cm–1M–1)).
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 solution.
23. 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) 76:381, Prog Histochem Cytochem (1976) 9:1)
24. M6494 also has Abs = 242 nm (EC = 21,000 cm–1M–1) in MeOH.
25. Generates chemiluminescence (Em = 465 nm in 0.1 M SDS) upon reaction with 1O2. (J Am Chem Soc (1986) 108:4498)
26. Generates chemiluminescence (Em = 455 nm) upon reaction with superoxide.
27. Spectroscopic properties of the product generated by reaction of M36008 with superoxide are described in Nat Protoc (2008) 3:8.
28. Oxidation of O13291 generates a fluorescent protein conjugate (Abs ~508 nm, Em ~528 nm).
29. Phycobiliproteins are packaged as suspensions in 60% ammonium sulfate, pH 7.0. Store refrigerated at 4°C but DO NOT FREEZE.
30. Iodoacetamides in solution undergo rapid photodecomposition to unreactive products. Minimize exposure to light prior to reaction.
31. Cis-parinaric acid is readily oxidized to nonfluorescent products. Use under N2 or Ar except when oxidation is intended. Stock solutions should be prepared in deoxygenated solvents.
Cis-parinaric acid is appreciably fluorescent in lipid environments and organic solvents but is nonfluorescent in water.
32. Acetate hydrolysis of R14000 yields rose bengal (Abs = 556 nm (EC = 104,000 cm–1M–1) Em = 572 nm in MeOH). (Photochem Photobiol (1997) 66:374)
33. R14060 is colorless and nonfluorescent until oxidized. The spectral characteristics of the oxidation product (2,3,4,5,6-pentafluorotetramethylrosamine) are similar to those of T639 (Section 12.2).
34. MW: The preceding ~ symbol indicates an approximate value, not including counterions.
35. The fluorescence of S36002 is relatively weak. Reaction of the dye with singlet oxygen (1O2) results in fluorescence enhancement with essentially no change in absorption or emission wave-
lengths.
36. Enzymatic reduction product is a water-soluble formazan, Abs = 475 nm.
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™
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822 IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to
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PRODUCT LIST 18.2 GENERATING AND DETECTING REACTIVE OXYGEN SPECIES

Cat. No. Product Quantity
A7923 4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethylpiperidin-1-oxyl, free radical (TEMPO-9-AC) 5 mg
A36003 3´-(p-aminophenyl) fluorescein (APF) *5 mM solution in DMF* 470 µL
A22188 Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit *500 assays* 1 kit
A12222 Amplex® Red reagent 5 mg
A22177 Amplex® Red reagent *packaged for high-throughput screening* 10 x 10 mg
A22182 Amplex® Red Xanthine/Xanthine Oxidase Assay Kit *400 assays* 1 kit
A36006 Amplex® UltraRed reagent 5 x 1 mg
B3932 (E,E)-3,5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY® 665/676) 5 mg
C400 5-(and-6)-carboxy-2´,7´-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) *mixed isomers* 25 mg
C2938 6-carboxy-2´,7´-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) 5 mg
C13293 5-(and-6)-carboxy-2´,7´-difluorodihydrofluorescein diacetate (carboxy-H2DFFDA) *mixed isomers* 5 mg
C7924 5-(2-carboxyphenyl)-5-hydroxy-1-((2,2,5,5-tetramethyl-1-oxypyrrolidin-3-yl)methyl)-3-phenyl-2-pyrrolin-4-one, potassium salt (proxyl fluorescamine) 5 mg
C6827 5-(and-6)-chloromethyl-2´,7´-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) *mixed isomers* *special packaging* 20 x 50 µg
C2944 coelenterazine 250 µg
D399 2´,7´-dichlorodihydrofluorescein diacetate (2´,7´-dichlorofluorescin diacetate; H2DCFDA) 100 mg
D2935 2´,7´-dichlorodihydrofluorescein diacetate, succinimidyl ester (OxyBURST® Green H2DCFDA, SE) 5 mg
D3861 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY® 581/591 C11) 1 mg
D23805 dihydrocalcein, AM *special packaging* 20 x 50 µg
D1168 dihydroethidium (hydroethidine) 25 mg
D23107 dihydroethidium (hydroethidine) *5 mM stabilized solution in DMSO* 1 mL
D11347 dihydroethidium (hydroethidine) *special packaging* 10 x 1 mg
D632 dihydrorhodamine 123 10 mg
D23806 dihydrorhodamine 123 *5 mM stabilized solution in DMSO* 1 mL
D633 dihydrorhodamine 6G 25 mg
D7894 diphenyl-1-pyrenylphosphine (DPPP) 5 mg
E33856 EnzChek® Myeloperoxidase (MPO) Activity Assay Kit *400 assays* *for myeloperoxidase chlorination and peroxidation activity* 1 kit
F2902 Fc OxyBURST® Green assay reagent *25 assays* *3 mg/mL* 500 µL
G36000 glutathione ethyl ester, biotin amide (BioGEE) *glutathiolation detection reagent* *special packaging* 10 x 100 µg
H36004 3´-(p-hydroxyphenyl) fluorescein (HPF) *5 mM solution in DMF* 470 µL
H7476 hypericin 1 mg
I36007 Image-iT® LIVE Green Reactive Oxygen Species Detection Kit *for microscopy* 1 kit
L6868 lucigenin (bis-N-methylacridinium nitrate) *high purity* 10 mg
L8455 luminol (3-aminophthalhydrazide) 25 g
M689 malachite green isothiocyanate 10 mg
M7913 trans-1-(2´-methoxyvinyl)pyrene 1 mg
M23800 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo1,2-apyrazin-3-one, hydrochloride (MCLA) 5 mg
M24571 merocyanine 540 25 mg
M36008 MitoSOX™ Red mitochondrial superoxide indicator *for live-cell imaging* 10 x 50 µg
M7511 MitoTracker® Orange CM-H2TMRos *special packaging* 20 x 50 µg
M7513 MitoTracker® Red CM-H2XRos *special packaging* 20 x 50 µg
M6494 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 1g
N6495 nitro blue tetrazolium chloride (NBT) 1g
O13291 OxyBURST® Green H2HFF BSA *special packaging* 5 x 1 mg
P36005 cis-parinaric acid *3 mM in ethanol* 10 mL
P6879 N-(1,10-phenanthrolin-5-yl)iodoacetamide 5 mg
P800 B-phycoerythrin *4 mg/mL* 0.5 mL
P801 R-phycoerythrin *4 mg/mL* 0.5 mL
R14060 RedoxSensor™ Red CC-1 *special packaging* 10 x 50 µg
R14000 rose bengal diacetate 5 mg
S36002 Singlet Oxygen Sensor Green *special packaging* 10 x 100 µg
X6493 XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) 100 mg
Z33857 Zen™ Myeloperoxidase (MPO) ELISA Kit *200 assays* 1 kit
The
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Probes®
™
Handbook: A Guide
A Guide Probes
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Chapter 18 — Probes for Reactive Oxygen Species, Including Nitric Oxide Section 18.3 Probes for Nitric Oxide Research
18.3 Probes for Nitric Oxide Research

Nitric oxide (NO) plays a critical role as a molecular mediator of a Carboxy-PTIO: A Nitric Oxide Antagonist
variety of physiological processes, including blood-pressure regulation Carboxy-PTIO (C7912) is a water-soluble and stable free radical
and neurotransmission.1–8 In endothelial cells, as well as in neurons and molecule that reacts stoichiometrically with NO.35–37 Carboxy-PTIO
astrocytes, NO is synthesized from L-arginine in a reaction catalyzed can be used in vivo to inhibit the physiological effects mediated by
by nitric oxide synthase (NOS) 9–12 (Figure 18.3.1). NO that diffuses into NO 35,37,38 or to quantitate NO levels in vitro by ESR spectrometry.39,40
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 SNAP: A Photoactivatable
largely on studies of NOS. Preparing NO solutions and detecting NO Nitric Oxide Donor
in experimental systems require special precautions to achieve repro-
ducibility.13,14 NO also reacts at diffusion-controlled rates with super- SNAP (S-nitroso-N-acetylpenicillamine; N7892, N7927) has been
oxide to form a strong oxidant, peroxynitrite anion 15 (ONOO –, Table shown to release nitric oxide (NO) in response to light stimulation in
18.1). Peroxynitrite is a well-known inflammatory mediator in various both aqueous and isopropyl alcohol solutions.41 The potential spatial
cardiovascular pathologies but has more recently been recognized as a and temporal control of NO release made possible by photolysis of NO
modulator of signal transduction pathways due to its ability to nitrate precursors makes this an attractive approach for generating NO in ex-
tyrosine residues and thereby influence cellular processes dependent on perimental systems.
tyrosine phosphorylation.15,16 Activated macrophages and neutrophils
produce nitric oxide and superoxide, and thus peroxynitrite anion, at
similar rates.17 NO generators are also reported to produce an accu- Detecting Nitric Oxide, Nitrite and Nitrate
mulation of chelatable Zn2+ in hippocampal neuronal perikarya, as de-
termined with some of our Zn2+ indicators 18 (Section 19.7, Table 19.6). The nitric oxide (NO) radical is short-lived and physiological
concentrations are very low,42 making in situ detection a challenging
proposition. NO is readily oxidized to the nitrosonium cation (NO+),
Spontaneous Nitric Oxide which is moderately stable in aqueous solutions but highly reactive with
Donors and Antagonist nucleophiles or other nitrogen oxides. Under aerobic conditions, these
reactive nitrogen oxides (Table 18.1) can be trapped by various amines,
Spermine NONOate in particular by aromatic amines to form diazonium salts or by aro-
Spermine NONOate (S7916) solids provide a means of preparing matic 1,2-diamines to form benzotriazoles (Figure 18.3.3).
aqueous NO solutions.19 When dissolved in buffer, cell culture medium
or blood, spermine NONOate dissociates to form two molecules of NO DAF-FM Nitric Oxide Indicator
and one molecule of the corresponding amine 20 (Figure 18.3.2). The First described in 1998,43 vicinal diamine derivatives of fluorescein
delivery of NO can be easily controlled by preparing moderately ba- generate stronger fluorescence signals at longer wavelengths than pro-
sic solutions of this NONOate and then lowering the pH to initiate totypes such as 2,3-diaminonaphthalene. These characteristics result
NO generation. Spermine NONOate releases NO slowly (half-life of in much enhanced performance for in situ nitric oxide detection. DAF-
39 minutes at 37°C in pH 7.4 buffer), making it suitable for whole ani- FM (4-amino-5-methylamino-2´,7´-difluorofluorescein) is the foremost
mal infusions and experiments with long incubations, 21 as well as for example of this class of compounds.44 We offer DAF-FM (D23841) and
in situ calibration of DAF-FM 22. its cell-permeant diacetate derivative (D23842, D23844). Like dihydro-
fluoresein, dihydrorhodamine and dihydroethidium probes (Section
SNAP and SIN-1 18.2), and in contrast to BAPTA-based Ca 2+ indicators (Section 19.2,
NO donors SNAP (S-nitroso-N-acetylpenicillamine; N7892, Section 19.3), DAF-FM is an endpoint dosimeter. DAF-FM is not a re-
N7927) and SIN-1 (3-morpholinosydnonimine, hydrochloride; versible equilibrium sensor, limiting its ability to track rapid fluctua-
M7891, M7926) spontaneously release NO (and superoxide in the tions of the target analyte (NO) in real time. Extracellularly applied
case of SIN-1) under physiological conditions (Figure 18.3.2), thereby DAF-FM diacetate spontaneously crosses the plasma membrane and
stimulating cyclic GMP production. 23–27 SNAP and SIN-1 have been is cleaved by esterases to generate intracellular DAF-FM, which is then
shown to be potent vasodilators in vivo and in vitro and to inhibit oxidized by NO to a triazole product accompanied by increased fluo-
smooth muscle cell mitogenesis and proliferation. 28–31 The relation- rescence (Figure 18.3.3, Figure 18.3.4). The fluorescence quantum yield
ship between NO generated from SNAP and SIN-1 and intracellular of DAF-FM is reported to be 0.005 but increases about 160-fold to 0.81
Ca 2+ has been studied using fluorescent Ca 2+ indicators 32–34 (Chapter after reacting with NO.44 The second step of the process as depicted
19). It has also been reported that NO released from SNAP stimulates in Figure 18.3.3 is an oversimplification. In fact, DAF-FM must first
Ca 2+-independent synaptic vesicle release, 34 which can be detected be nonspecifically oxidized to an anilinyl radical, which then reacts
with FM® 1–43 (T3163, T35356; Section 16.1). We offer SNAP and with NO to form the fluorescent triazole product.45 This mechanistic
SIN-1 in 25 mg vials (N7892, M7891) and, because of their potential complication must be borne in mind when interpreting experimental
instability in solution, as specially packaged sets of 20 vials, each con- data. Specifically, the question of whether nonspecific pre-oxidation or
taining 1 mg (N7927, M7926). reaction with NO is the dominant factor controlling observed DAF-FM
The
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in thisaremanual
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Appendix onto
824 IMPORTANT NOTICE : The products described are by one or more Limited Use Label License(s).
fluorescence signals requires critical scrutiny.45–47 Applications of DAF-FM and DAF-FM diac-
1.2 µM NO
etate include:
1.1
0.89
48
• Assessment of NO production in transaldolase-deficient lymphoblasts by flow cytometry 0.71
• Detection of NO accumulation in embryonic cortical neurons following neurotrophin 0.54

0.36
stimulation 49 0.18
• In vivo imaging of NO in zebrafish 50 0
• Intravital microscopic detection of NO generation associated with angiogenesis in mice 51

• Quantitation of ATP-induced NO release in rabbit platelets 22
475 500 525 550 575 600
2,3-Diaminonaphthalene Wavelength (nm)
In a reaction similar to that of DAF-FM (Figure 18.3.3), 2,3-diaminonaphthalene (D7918, Figure 18.3.4 Fluorescence emission spectra of DAF-FM
Figure 18.3.5) reacts with the nitrosonium cation that forms spontaneously from NO to form the (D23841, D23842) in solutions containing 0 to 1.2 µM nitric
fluorescent product 1H-naphthotriazole.52,53 Using 2,3-diaminonaphthalene, researchers have oxide (NO).
developed a rapid, quantitative fluorometric assay that can detect from 10 nM to 10 µM nitrite
and is compatible with a 96-well microplate format.54
NH2
NH2 NH2
+
C NH2 C O
NH2
NH NADPH NADP + NH
Figure 18.3.5 2,3-diaminonaphthalene (D7918).
)CH2) 3
+ O2 (C H2) 3
+ NO
HOOC CH HOOC CH
+NH NO Synthase +NH
3 3
L-Arginine L-Citrulline
Figure 18.3.1 Nitric oxide synthase production of nitric oxide (NO) and L-citrulline from L-arginine and dioxygen (O2).
A _ B
O H+
RN N O RH + 2 NO 2R S N O RSSR + 2 NO
C
NH
_ + +
O N N +
_ + OH O N N CH2CN O N N CHCN + NO + H
N O N O
_
O2 O2
Figure 18.3.2 Mechanisms of spontaneous NO release by: A) Spermine NONOate (S7916); B) SNAP (N7892, N7927); and C) SIN-1 (M7891, M7926).
Cell membrane
O O
< <
CH C O O O C CH O O O O O O
3 3
F O F Esterase F F F F
< NO <
O C O C O
O O
NH NH N
2 2
NHCH NHCH N N
3 3 H C
3
DAF-FM diacetate DAF-FM Benzotriazole derivative

(Nonfluorescent, cell-permeant) (Weakly fluorescent) (Fluorescent)
Figure 18.3.3 Reaction scheme for the detection of nitric oxide (NO) by DAF-FM (D23841) and DAF-FM diacetate (D23842, D23844).
The
TheMolecular
Probes®
™
A Guide
A Guide Probes
to Fluorescent and
Probes Labeling
and Technologies
manualinare
thiscovered
manual are
by covered by one or moreUse
Limited
LabelUse Label License(s).
PleasePlease
refer refer to Appendix
the Appendix
on on
IMPORTANT NOTICE : The products in this one or more Limited License(s). to the
1,2-Diaminoanthraquinone
For directly detecting NO levels in vivo, we offer 1,2-diaminoanthraquinone (DAA,
D23840). This nitric oxide probe is reported to be nonfluorescent until it reacts with NO to pro-
duce a red-fluorescent precipitate. 1,2-Diaminoanthraquinone has been used to detect changes in
NO levels in rat retinas after injury to the optic nerve.55 This methodology may make it possible
to test the actions of NO in neurodegeneration, inflammation and other biological processes. The
role of NO production in hippocampal long-term potentiation has also been investigated using
1,2-diaminoanthraquinone for spatial imaging of NO in rat brain slices.56,57
NBD Methylhydrazine
NBD methylhydrazine (N-methyl-4-hydrazino-7-nitrobenzofurazan, M20490) is a unique
reagent for the detection of nitrite. Reaction of NBD methylhydrazine with NO2– in the presence
of mineral acids leads to formation of fluorescent products with excitation/emission maxima of
~468/537 nm. This reaction serves as the principle behind a selective fluorogenic method for
the determination of NO2– (Figure 18.3.6). Although NBD methylhydrazine has been used to
quantitate nitrite in water using a fluorescence microplate reader, 58 it does not seem to have been
used yet to detect nitrite formed by spontaneous oxidation of NO.
Dichlorodihydrofluorescein Diacetate and Dihydrorhodamine 123

In addition to their extensive use for detecting other reactive oxygen species such as su-
peroxide, dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydrorhodamine 123 (D399,
D632; Section 18.2) have been reported to be useful for detecting peroxynitrite formation both
in solution and in live cells.59
Figure 18.3.7 Fixed and permeabilized bovine pulmonary
artery endothelial cells were treated with either degraded
peroxynitrite (top panel) or 100 µM peroxynitrite (bottom Anti-Nitrotyrosine Antibody
panel) for 5 minutes at room temperature to induce protein High levels of nitrotyrosine are associated with a large number of diseases, including mul-
nitration. Nitrated tyrosine residues were detected with our
rabbit anti-nitrotyrosine antibody (A21285) and visualized tiple sclerosis, Alzheimer disease and Parkinson disease.60–62 Increased levels of nitrotyrosine
with the green-fluorescent Alexa Fluor® 488 goat anti–rab- are also indicative of vascular and tissue injury from ischemia–reperfusion and inflammation.61
bit IgG antibody (A11008). Nuclei were counterstained with Several pathways for the nitration of tyrosine have been suggested. Peroxynitrite (OONO –),
blue-fluorescent DAPI (D1306, D3571, D21490).
formed by spontaneous reaction of nitric oxide (NO) with superoxide (•O2–), elicits downstream
tyrosine nitration.61,63 Heme peroxidases, such as myeloperoxidase and eosinophil peroxidase,
have been shown to utilize hydrogen peroxide (H2O2) to oxidize nitrite (NO2–) and catalyze
tyrosine nitration.64 In addition, other heme proteins such as hemoglobin and catalase may con-
tribute to tyrosine nitration using NO as a substrate.65 Tryptophan residues can also be oxidized
by peroxynitrite.66
We offer a high-activity rabbit polyclonal anti-nitrotyrosine antibody (A21285) for detecting
nitrotyrosine-containing proteins and peptides. This antibody is suitable for both immunohis-
tochemical (Figure 18.3.7) and western blotting (Figure 18.3.8) applications and is useful for
CaptAvidin™ Dimer
identifying nitrated proteins and determining the level of protein nitrosylation in tissues.67,68
Fluorescence of Green Fluorescent Protein (GFP) is extremely sensitive to tyrosine nitration, as
CaptAvidin™
confirmed by correlated anti-nitrotyrosine immunoreactivity.69
S-Nitrosothiol Detection
1 2 S-nitrosylation of thiols, principally in the form of cysteine sidechains or glutathione, is a
primary mechanism for downstream propagation of nitric oxide release events. This reversible
Figure 18.3.8 Specificity of our rabbit anti-nitrotyrosine an-
posttranslational modification regulates enzymatic activity, subcellular localization, chromatin
tibody (A21285) to nitrated proteins. Equal amounts of avi-
din (A887, lane 1) and CaptAvidin™ biotin-binding protein remodeling and protein degradation.49,70 The primary reactive nitrogen species responsible for
(C21385, lane 2) were run on an SDS-polyacrylamide gel
(4–20%) and blotted onto a PVDF membrane. CaptAvidin™
biotin-binding protein, a derivative of avidin, has nitrated
tyrosine residues in the biotin-binding site. On a western
H C N NH H C NH
blot, nitrated proteins were identified with the anti-nitroty- 3 2 3
— +
rosine antibody in combination with an alkaline phospha- N NO H N
2
tase conjugate of goat anti–rabbit IgG antibody (G21079)
and the red-fluorescent substrate DDAO phosphate (D6487). O O
N N
NO NO
2 2
Figure 18.3.6 Reaction scheme illustrating the principle of nitrite detection by NBD methylhydrazine (M20490).
The
Handbook: A
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™
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Appendix on to
S-nitrosylation of protein thiols is dinitrogen dioxide (N2O3) formed be used to analyze NO that has been trapped as an S-nitroso derivative
from O2 and NO. Techniques for detecting S-nitrosothiol modifica- by a modification that uses mercuric chloride or copper (II) acetate to
tions exploit, but are also compromised by, their reversible nature and release the NO from its complex.83,84
their susceptibility to photolytic cleavage. The technique with most
widespread adoption, often referred to as the biotin switch method,71–73 Measure-iT™ High-Sensitivity Nitrite Assay Kit
consists of three steps: (1) blocking of free thiols with N-ethylmaleimide The Measure-iT™ High-Sensitivity Nitrite Assay Kit (M36051) pro-
or another alkylating reagent, (2) selective reduction of S-nitrosothiols vides an easy and accurate method for quantitating nitrite. This kit has
to thiols using ascorbate or TCEP (T2556, Section 2.1) and (3) labeling an optimal range of 20–500 picomoles nitrite (Figure 18.3.10), making
of thiols created in step 2 with a fluorescent or biotinylated maleimide it up to 50 times more sensitive than colorimetric methods utilizing
or iodoacetamide reagent 74,75 (Section 2.2 or Section 4.2, respective- the Griess reagent. Nitrates may be analyzed after quantitative conver-
ly). Streptavidin agarose (S951, Section 7.6) can be used to subsequently sion to nitrites through enzymatic reduction; 80 used in this manner, the
pull down biotinylated proteins for further analysis if required. The over- Measure-iT™ nitrite assay also provides an effective method for quan-
all technique is vulnerable to false positives through incomplete block- titating nitric oxide.
ing of unmodified thiols in step 1 and inadvertent reduction of disulfides Each Measure-iT™ High-Sensitivity Nitrite Assay Kit contains:
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- • Measure-iT™ nitrite quantitation reagent (100X concentrate in
traviolet light, releasing NO and subsequently nitrite (NO2–).76 The NO 0.62 M HCl)
product of this process can be detected using DAF-FM 77 or the nascent • Measure-iT™ nitrite quantitation developer (2.8 M NaOH)
thiol product can be detected using a fluorescent maleimide reagent.78 • Measure-iT™ nitrite quantitation standard (11 mM sodium nitrite)
Griess Reagent Kit
Under physiological conditions, NO is readily oxidized to nitrite Simply dilute the reagent 1:100, load 100 µL into the wells of a
and nitrate or it is trapped by thiols as an S-nitroso adduct. The Griess microplate, add 1–10 µL sample volumes and mix. After a 10-minute
reagent provides a simple and well characterized colorimetric assay for incubation at room temperature, add 5 µL of developer and read the
nitrites, and nitrates that have been reduced to nitrites, with a detection fluorescence. The assay signal is stable for at least 3 hours, and com-
limit of about 100 nM.52,79,80 Nitrites react with sulfanilic acid in acidic mon contaminants are well tolerated in the assay. The Measure-iT™
solution to form an intermediate diazonium salt that couples to N-(1- High-Sensitivity Nitrite Assay Kit provides sufficient material for 2000
naphthyl)ethylenediamine to yield a purple azo derivative that can be assays, based on a 100 µL assay volume in a 96-well microplate format;
monitored by absorbance at 548 nm (Figure 18.3.9). this nitrite assay can also be adapted for use in cuvettes or 384-well
Our Griess Reagent Kit (G7921) contains all of the reagents re- microplates.
quired for nitrite quantitation, including:
• N-(1-Naphthyl)ethylenediamine dihydrochloride
• Sulfanilic acid in 5% H3PO4
R2 = 0.9995
• Concentrated nitrite quantitation standard for generating calibra-
tion curves
Fluorescence
• 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 0 100 200 300 400 500
to reduce nitrate without producing excess NADPH, which can inter- Nitrite (pmol)
fere with the Griess reaction.81 A review of the use of the Griess re- Figure 18.3.10 Linearity and sensitivity of the Measure-iT™ high-sensitivity nitrite as-
agent for nitrite and nitrate quantitation in human plasma describes say. Triplicate 10 µL samples of nitrite were assayed using the Measure-iT™ High-Sensitivity
Nitrite Assay Kit (M36051). Fluorescence was measured using excitation/emission of
optimal reaction conditions for minimizing interference from plasma 365/450 nm and plotted versus picomoles of nitrite. Background fluorescence was not sub-
constituents (particularly NADPH).82 The Griess Reagent Kit can also tracted. The variation (CV) of replicate samples was <2%.
_
NO2
+ NH(CH ) NH
HO3S NH2 HO3S N2 + 22 2 HO S N N NH(CH ) NH
3 22 2
Diazonium salt Azo dye

Figure 18.3.9 Principle of nitrite quantitation using the Griess Reagent Kit (G7921). Formation of the azo dye is detected via
its absorbance at 548 nm.
TheMolecular
Probes Handbook:
A Guide Probes
to Fluorescent and
Probes Labeling
and Technologies
™
manualinare
thiscovered
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or moreUse
PleasePlease
on on
REFERENCES
1. Science (2001) 292:2413; 2. Science (2001) 292:2486; 3. Curr Biol (1997) 7:R376; 4. Cell Radic Biol Med (2007) 43:995; 46. Anal Chem (2006) 78:1859; 47. Free Radic Biol Med
(1994) 78:919; 5. Am J Physiol (1992) 262:G379; 6. Annu Rev Biochem (1994) 63:175; (2005) 39:327; 48. Biochem J (2008) 415:123; 49. Nature (2008) 455:411; 50. Free Radic
7. J Med Chem (1995) 38:4343; 8. Science (1992) 257:494; 9. Biochem J (2001) 357:593; Biol Med (2007) 43:619; 51. Methods Enzymol (2008) 441:393; 52. Luminescence
10. Biochem J (1994) 298:249; 11. Cell (1994) 78:915; 12. J Med Chem (1994) 37:1899; (1999) 14:283; 53. Methods Enzymol (1996) 268:105; 54. Anal Biochem (1993) 214:11;
13. Methods Mol Biol (1998) 100:215; 14. Curr Opin Chem Biol (2010) 14:43; 15. Free 55. Neuroreport (1998) 9:4051; 56. Neurobiol Dis (2002) 11:96; 57. Neuroimage (2002)
Radic Biol Med (2001) 30:463; 16. Front Biosci (2009) 14:4809; 17. Methods Enzymol 15:633; 58. Anal Chem (1999) 71:3003; 59. Methods Enzymol (2008) 441:261; 60. J Biol
(1994) 233:229; 18. Brain Res (1998) 799:118; 19. Methods Enzymol (1996) 268:281; Chem (2003) 278:8380; 61. Free Radic Res (2000) 33:771; 62. Methods Enzymol
20. J Med Chem (1991) 34:3242; 21. J Biol Chem (2001) 276:28799; 22. Anal Chem (1999) 301:373; 63. Proc Natl Acad Sci U S A (1996) 93:11853; 64. Biochem Biophys
(2007) 79:2421; 23. Nature (1993) 364:626; 24. Nature (1995) 375:68; 25. Brain Res (1993) Res Commun (2001) 285:273; 65. Biochim Biophys Acta (2001) 1528:97; 66. Biochem
619:344; 26. FEBS Lett (1993) 315:139; 27. Thromb Res (1993) 70:405; 28. J Pharmacol Biophys Res Commun (1997) 234:82; 67. Nat Clin Pract Cardiovasc Med (2008) 5:811;
Exp Ther (1992) 260:286; 29. Eur J Pharmacol (1987) 144:379; 30. J Pharmacol Exp 68. Methods Mol Biol (2008) 477:41; 69. Proc Natl Acad Sci U S A (2002) 99:3481;
Ther (1989) 248:762; 31. J Clin Invest (1989) 83:1774; 32. Am J Physiol (1994) 266:L9; 70. Proc Natl Acad Sci U S A (2005) 102:117; 71. Free Radic Biol Med (2009) 46:119;
33. Life Sci (1994) 54:1449; 34. Neuron (1994) 12:1235; 35. Biochemistry (1995) 34:7177; 72. Proteomics (2009) 9:808; 73. Methods Enzymol (2008) 441:53; 74. Nitric Oxide
36. Biochem Biophys Res Commun (1994) 202:923; 37. Biochemistry (1993) 32:827; (2008) 19:295; 75. J Bacteriol (2008) 190:4997; 76. J Biol Chem (1996) 271:18596; 77. Anal
38. Infect Immun (1993) 61:3552; 39. Photochem Photobiol (1995) 61:325; 40. Life Sci Biochem (2005) 346:69; 78. J Biol Chem (2006) 281:40354; 79. FASEB J (1993) 7:349;
(1994) 54:185; 41. Photochem Photobiol (1998) 67:282; 42. Nitric Oxide (2009) 21:92; 80. Anal Biochem (1982) 126:131; 81. Anal Biochem (1995) 224:502; 82. Methods
43. Anal Chem (1998) 70:2446; 44. Angew Chem Int Ed Engl (1999) 38:3209; 45. Free Enzymol (2008) 440:361; 83. Anal Biochem (1996) 238:150; 84. Chem Biol (1996) 3:655.
DATA TABLE 18.3 PROBES FOR NITRIC OXIDE RESEARCH

C7912 315.39 FF,D H2O 367 9300 none MeOH
D7915 155.13 FF,DD,A H2O, DMSO 248 8000 none pH 12 1
D7918 158.20 L DMSO, MeOH 340 5100 377 MeOH 2
D23840 336.32 F,D,L DMSO 521 6000 none MeOH 3
D23841 412.35 F,D,L DMSO 487 84,000 see Notes pH 8 4
D23842 496.42 F,D,L DMSO <300 none 5
D23844 496.42 F,D,L DMSO <300 none 5
M7891 206.63 FF,D,LL DMSO, H2O 291 11,000 none pH 7 6
M7926 206.63 FF,D,LL DMSO, H2O 291 11,000 none pH 7 6
M20490 209.16 F,L MeCN 487 24,000 none MeOH 7
N7892 220.24 FF,D,LL DMSO, H2O 342 700 none MeOH 6
N7927 220.24 FF,D,LL DMSO, H2O 342 700 none MeOH 6
S7916 262.35 FF,DD,A H2O, DMSO 248 8200 none pH 12 1
For definitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.
Notes
1. Releases nitric oxide upon acid-catalyzed dissociation in solution. Stable in alkaline solutions. (Methods Enzymol (1996) 268:281)
2. Fluorescence of D7918 is weak. Reaction with nitrite yields highly fluorescent 1H-naphthotriazole (Abs = 365 nm, Em = 415 nm in H2O (pH 12)). (Methods Enzymol (1996) 268:105)
3. 1,2-Diaminoanthraquinone reacts with nitrite or nitric oxide to produce 1H-anthratriazole-6,11-dione which forms a red-fluorescent (Em >580 nm) precipitate in water. (Neuroreport (1998) 9:4051)
4. DAF-FM fluorescence is very weak. Reaction with nitrite or nitric oxide generates a highly fluorescent benzotriazole derivative with Abs = 495 nm (EC = 73,000 cm–1M–1), Em = 515 nm in pH 7.4
buffer. (Angew Chem Int Ed Engl (1999) 38:3209)
5. Acetate hydrolysis and subsequent reaction with nitrite or nitric oxide generate a highly fluorescent benzotriazole derivative with Abs = 495 nm (EC = 73,000 cm–1M–1), Em = 515 nm in pH 7.4
buffer. (Angew Chem Int Ed Engl (1999) 38:3209)
6. Spontaneously decomposes in solution.
7. NBD methylhydrazine reacts with nitrite in the presence of strong acid to form fluorescent N-methyl-4-amino-7-nitrobenzofurazan (Abs = 459 nm, Em = 537 nm in MeCN). (Anal Chem (1999)
71:3003)
PRODUCT LIST 18.3 PROBES FOR NITRIC OXIDE RESEARCH

Cat. No. Product Quantity
A21285 anti-nitrotyrosine, rabbit IgG fraction *1 mg/mL* 0.5 mL
C7912 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, potassium salt (carboxy-PTIO) 25 mg
D23841 DAF-FM (4-amino-5-methylamino-2´,7´-difluorofluorescein) 1 mg
D23842 DAF-FM diacetate (4-amino-5-methylamino-2´,7´-difluorofluorescein diacetate) 1 mg
D23844 DAF-FM diacetate (4-amino-5-methylamino-2´,7´-difluorofluorescein diacetate) *special packaging* 10 x 50 µg
D23840 1,2-diaminoanthraquinone sulfate (DAA) *high purity* 5 mg
D7918 2,3-diaminonaphthalene 100 mg
G7921 Griess Reagent Kit *for nitrite quantitation* 1 kit
M36051 Measure-iT™ High-Sensitivity Nitrite Assay Kit *2000 assays* 1 kit
M20490 N-methyl-4-hydrazino-7-nitrobenzofurazan (NBD methylhydrazine) 25 mg
M7891 3-morpholinosydnonimine, hydrochloride (SIN-1) 25 mg
M7926 3-morpholinosydnonimine, hydrochloride (SIN-1) *special packaging* 20 x 1 mg
N7892 S-nitroso-N-acetylpenicillamine (SNAP) 25 mg
N7927 S-nitroso-N-acetylpenicillamine (SNAP) *special packaging* 20 x 1 mg
S7916 spermine NONOate 10 mg
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CH 18 Reactive Oxygen Species

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The Molecular Probes® Handbook

A GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES

Molecular Probes™ Handbook

Molecular Probes Resources

Molecular Molecular Probes®SpectraViewer

BioProbes® Journal of Cell Biology Applications

Access all Molecular Probes® educational resources at lifetechnologies.com/mpeducat

18.2 Generating and Detecting Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806

Dihydrorhodamine 123 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 818

18.3 Probes for Nitric Oxide Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 824

18.1 Introduction to Reactive Oxygen Species

Table 18.1 Reactive oxygen species.

18.2 Generating and Detecting Reactive Oxygen Species

IMPORTANT NOTICE: The products described in this manual

Detecting Singlet Oxygen

Generating Hydroxyl and Superoxide Radicals

peroxide. Thus, fluorescence excitation at 400 nm with emission detec-

transporter activity, measured using the aminostyryl dye substrate

4-Di-1-ASP (D288, Section 12.2), has been investigated in mouse

focal microscopy analysis of reactive oxygen species (ROS) produc-

oxidation in resting leukocytes, possibly through the uncoupling of mitochondrial oxidative

Table 18.2 Scavengers of reactive oxygen species (ROS). H3C N CH3

idative events in cells rich in peroxidases, including granulocytes 129–132 N N

Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit

Amplex® Red Xanthine/Xanthine Oxidase Assay Kit

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

• Amplex® Red reagent • Xanthine oxidase from buttermilk

EnzChek® Myeloperoxidase (MPO) Activity Assay Kit

100 Figure 18.2.25 Detection of hypoxanthine using the

The EnzChek® Myeloperoxidase (MPO) Activity Assay Kit (E33856) pro-

Sufficient reagents are provided to perform 200 assays for chlorination

prehensive set of components for accurate and sensitive quantitation of hu-

100 • Amplex® UltraRed reagent

Assaying Oxidative Activity in Live Cells 1,000

Figure 18.2.32 5-(and-6)-chloromethyl-2’,7’-dichlorodihydro-

of cells labeled with 5-(and 6-)chloromethyl-2’,7’-dichlorodihydroflu-

Image-iT® LIVE Green Reactive Oxygen Species Detection Kit

• Carboxy-H2DCFDA (5-(and 6-)carboxy-2´,7´-dichlorodihydrofluorescein diacetate)

This assay is based on carboxy-H2DCFDA (5-(and 6-)carboxy-2´,7´-dichlorodihydrofluorescein

Aminophenyl Fluorescein and Hydroxyphenyl Fluorescein

Amine-Reactive OxyBURST® Green Reagent

A Longer-Wavelength Reduced Rhodamine Figure 18.2.40 Rhodamine 123 (R302).

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

Reduced MitoTracker® Probes

Figure 18.2.43 RedoxSensor™ Red CC-1 (R14060).

permeabilized, glutathiolation levels can be detected with a fluorescent streptavidin conjugate

IMPORTANT NOTICE: The products described in this manual

DATA TABLE 18.2 GENERATING AND DETECTING REACTIVE OXYGEN SPECIES

DATA TABLE 18.2 GENERATING AND DETECTING REACTIVE OXYGEN SPECIES—continued

PRODUCT LIST 18.2 GENERATING AND DETECTING REACTIVE OXYGEN SPECIES

18.3 Probes for Nitric Oxide Research

IMPORTANT NOTICE: The products described in this manual

• Detection of NO accumulation in embryonic cortical neurons following neurotrophin 0.54

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

DAF-FM diacetate DAF-FM Benzotriazole derivative

Dichlorodihydrofluorescein Diacetate and Dihydrorhodamine 123

• Detailed protocols for spectrophotometer and microplate reader

Both the N-(1-naphthyl)ethylenediamine dihydrochloride and the

Diazonium salt Azo dye

DATA TABLE 18.3 PROBES FOR NITRIC OXIDE RESEARCH

PRODUCT LIST 18.3 PROBES FOR NITRIC OXIDE RESEARCH

IMPORTANT NOTICE: The products described in this manual

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