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Peroxynitrite/Scavengers/Detection
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ALX-620-075 Revised 24-Jan-08
APF
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SYNONYMS Aminophenyl fluorescein
2-[6-(4'-Amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid
PRODUCT LINE Oxidative Stress
PRODUCT CATEGORY Oxidative Stress Markers & Reagents/Related Products
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Product Numbers: Format: Size: Unit Price: Quantity: Add To Cart
ALX-620-075-M001   1 mg 430.00 USD Add To Cart
Product Specification
FORMULA: C26H17NO5
MW: 423.4
CAS NUMBER: 359010-70-1
CONCENTRATION: ~5mM
PURITY: ≥98% (HPLC)
FORMULATION: Liquid. In 0.47ml dimethyl formamide.
SHIPPING: AMBIENT
LONG TERM STORAGE: +4°C
USE/STABILITY: Prepare 500-5’000-fold dilution (~10-1µM) in phosphate buffer (0.1M phosphate, pH 7.4) immediately before use. BSA, phenol red and amines may affect the fluorescence and must be used with caution. Do not store the dilutions.
HANDLING: Keep under inert gas. Protect from light. After opening, prepare aliquots and store at +4°C.
HAZARD: HARMFUL.

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Product Description
Fluorescent reagent (Ex(max): 490nm; Em(max): 515nm) for the detection of highly reactive oxygen species (hROS). Immediately reacts with hROS such as hydroxyl radical, peroxynitrite and hypochlorite, and the fluorescence intensity greatly increases. Use of APF together with HPF (Prod. No. ALX-620-074) also allows for specific detection of hypochlorite  (-OCl) to elucidate reliable the roles of -OCl in biological systems such as neutrophils. In addition, peroxynitrite can be detected in distinction from nitric oxide and superoxide since APF does not react with nitric oxide, superoxide and hydrogen peroxide. Moreover, APF is resistant to light-induced autooxidation. Not for sale in Japan.
Product Specific Literature References
Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species: K. Setsukinai, et al.; J. Biol. Chem. 278, 3170 (2003) Abstract
General Information
BACKGROUND/TECHNICAL INFORMATION Precautions for use:
1. Prepare the diluted solution just before use and use it up.
2. The dilution buffer should be adjusted to pH 7.0-7.5. Please note that bovine serum albumin (BSA) and phenol red might interfere with the fluorescence measurement.

Light-induced autoxidation (Figure 3)
After adding HPF or DCFH-DA (10µM), the cells were incubated for 30 min at 37°C in the dark. The fluorescence images were obtained with a confocal fluorescent microscope (excitation wavelength: 488nm, fluorescence emission wavelength: using a 515nm barrier filter). The cells were laser-irradiated again at 488nm for 10 sec and fluorescence images were obtained again (see Figure 3).

Fluorescence images of HPF- or APF-loaded neutrophils (Figure 4)
Neutrophils were obtained from porcine blood and suspended in Krebs-Ringer phosphate buffer (114mM NaCI, 4.6mM KCI, 2.4mM MgSO4, 1.0mM CaCI2, 15mM NaH2PO4/Na2HPO4, pH 7.4) and kept on ice until use. Then they were seeded onto a glass-bottomed dish. The cells were loaded with HPF or APF (10µM) by incubation for 30min at room temperature and then stimulated with PMA (including 2ng/ml; 0.1% DMF as a cosolvent). Fluorescence images were obtained with a confocal fluorescent microscope before and 10 min after the stimulation with PMA (excitation wavelength: 488nm, fluorescence emission wavelength: 505-550nm; using a 505-550nm barrier filter).

Detection of hROS in the HRP/H2O2 system using HPF and APF (Figure 5)
Reaction timecourse
Fluorescence probe reagents (final 10µM; 0.1% DMF as a cosolvent) were added to sodium phosphate buffer (0.1M; pH 7.4) containing HRP (0.2µM). H2O2 (final 1µM) was added at the time indicated by the arrow. The fluorescence intensities were measured at excitation wavelength of 490nm and fluorescence emission wavelength of 515nm. Relation between the amount of added H2O2 and fluorescence increase in the HRP/H2O2 system.

Relation between the amount of added H2O2 and fluorescence increase in the HRP/H2O2 system
Fluorescence probe reagents (final 10µM; 0.1% DMF as a cosolvent) were added to sodium phosphate buffer (0.1M; pH 7.4) containing HRP (0.2µM). The fluorescence intensities were measured at excitation wavelength of 490nm and fluorescence emission wavelength of 515nm.
MANUFACTURER Manufactured by Daiichi Pure Chemicals Co. Ltd., Japan.
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Further Categories Containing This Product:
Peroxynitrite/Scavengers/Detection • Dyes/Stains/Fluorescent Probes/Fluorescent Labels
 
 
ALX-620-063 Revised 03-Feb-05
Coelenterazine
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SYNONYMS 2-[(4-Hydroxyphenyl)methyl]-6-(4-hydroxyphenyl)-8-(phenylmethyl)-imidazo [1,2-a] pyrazin-3-(7H)-one
PRODUCT LINE Oxidative Stress
PRODUCT CATEGORY Peroxynitrite/Scavengers/Detection
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ALX-620-063-C050   50 µg 110.00 USD Add To Cart
Product Specification
FORMULA: C26H21N3O3
MW: 423.5
CAS NUMBER: 55779-48-1
PURITY: ≥95%
APPEARANCE: Brown solid.
SOLUBILITY: Slightly soluble in methanol or ethanol. Avoid DMSO.
SHIPPING: SHIPPED ON BLUE ICE
LONG TERM STORAGE: -20°C
HANDLING: Protect from light. Keep under inert gas. Keep calcium free when stored in solution.

Product Description
Cell permeable, very sensitive and specific chemiluminescence probe of the superoxide anion and peroxynitrite. When oxidized by oxygen it emits blue light at 446nm when Ca2+ binds to the complex. Powerful antioxidant. See also 2-(4-Dehydroxy)coelenterazine (Prod. No. ALX-620-062).
Product Specific Literature References
Use of calcium-regulated photoproteins as intracellular Ca2+ indicators: J.R. Blinks; Meth. Enzymol. 172, 164 (1989) Abstract
Slow calcium waves accompany cytokinesis in medaka fish eggs: R.A. Fluck, et al.; J. Cell Biol. 115, 1259 (1991) Abstract
Coelenterazine is a superoxide anion-sensitive chemiluminescent probe: its usefulness in the assay of respiratory burst in neutrophils: M. Lucas & F. Solano; Anal. Biochem. 206, 273 (1992) Abstract
Aequorin-expressing mammalian cell lines used to report Ca2+ mobilization: D. Button & M. Brownstein; Cell Calcium 14, 663 (1993) Abstract
Imaging [Ca2+]i with aequorin using a photon imaging detector: A.L. Miller, et al.; Meth. Cell Biol. 40, 305 (1994) Abstract
Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyfish apoaequorin cDNA: S. Saran, et al.; FEBS Lett. 337, 43 (1994) Abstract
The origins of marine bioluminescence: turning oxygen defence mechanisms into deep-sea communication tools: J.F. Rees, et al.; J. Exp. Biol. 201, 1211 (1998) Abstract; Full Text
Chemiluminescent detection of oxidants in vascular tissue. Lucigenin but not coelenterazine enhances superoxide formation: M.M. Tarpey, et al.; Circ. Res. 84, 1203 (1999) Abstract
Further Categories Containing This Product:
Dyes/Stains/Fluorescent Probes/Fluorescent Labels • Oxidative Stress Markers & Reagents/Related Products • Antioxidants, Flavonoids & Free Radical Scavengers Other Products • Luciferin & Luciferase/Related Products • Calcium Indicators/Probes
 
 
ALX-620-062 Revised 01-Jun-06
2-(4-Dehydroxy)coelenterazine
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SYNONYMS Coelenterazine h
PRODUCT LINE Oxidative Stress
PRODUCT CATEGORY Peroxynitrite/Scavengers/Detection
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ALX-620-062-C050   50 µg 110.00 USD Add To Cart
Product Specification
FORMULA: C26H21N3O2
MW: 407.5
PURITY: ≥95%
APPEARANCE: Yellow to orange solid.
SOLUBILITY: Soluble in 100% ethanol or methanol; slightly soluble in water. Avoid DMSO as product is unstable in this solvent.
SHIPPING: SHIPPED ON BLUE ICE
LONG TERM STORAGE: -20°C
HANDLING: Protect from light. Keep under inert gas. Keep calcium free when stored in solution.
HAZARD: IRRITANT.

Product Description
Derivative of coelenterazine (Prod. No. ALX-620-063) with ~20 times higher luminescence intensity.
Product Specific Literature References
Semi-synthetic aequorins with improved sensitivity to Ca2+ ions: O. Shimomura, et al.; Biochem. J. 261, 913 (1989) Abstract
Preparation and handling of aequorin solutions for the measurement of cellular Ca2+: O. Shimomura; Cell Calcium 12, 635 (1991) Abstract
Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins: M.R. Knight, et al.; J. Cell. Biol. 121, 83 (1993) Abstract
Intracellular Ca2+ signals in Dictyostelium chemotaxis are mediated exclusively by Ca2+ influx: T. Nebl & P.R. Fisher; J. Cell Sci. 110, 2845 (1997) Abstract
Further Categories Containing This Product:
Dyes/Stains/Fluorescent Probes/Fluorescent Labels • Oxidative Stress Markers & Reagents/Related Products • Antioxidants, Flavonoids & Free Radical Scavengers Other Products • Calcium Indicators/Probes
 
 
ALX-610-022 Revised 25-Apr-08
2',7'-Dichlorodihydrofluorescein diacetate
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SYNONYMS DCDHF diacetate
2',7'-Dichlorofluorescein diacetate
PRODUCT LINE Other Products
PRODUCT CATEGORY Dyes/Stains/Fluorescent Probes/Fluorescent Labels
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ALX-610-022-M050   50 mg 43.00 USD Add To Cart
Product Specification
FORMULA: C24H16O7Cl2
MW: 487.3
CAS NUMBER: 4091-99-0
PURITY: ≥95%
APPEARANCE: White to off-white solid.
SOLUBILITY: 25mg/ml soluble in DMSO, 100% ethanol or dimethyl formamide; sparingly soluble in aqueous buffers.
SHIPPING: SHIPPED ON BLUE ICE
LONG TERM STORAGE: -20°C
USE/STABILITY: Stock solution in organic solvents is stable for at least 6 months if stored at -20°C. For maximum solubility in aqueous buffers, dissolve directly in 0.1M disodium carbonate (7.7mg/ml) and then immediately diluted with PBS, pH 7.2, to the desired concentration or pH. Upon neutralization of the solution the concentration may not be greater than 1mg/ml. We do not recommend storing the aqueous solution for more than one day.

Product Description
Cell permeable, sensitive indicator of peroxynitrite formation. After hydrolysis of the diacetate groups by cytosolic esterases or base-catalyzed cleavage of the diacetate groups, DCDHF is oxidized by peroxynitrite to the highly fluorescent product dichlorofluorescein (DCF). Formation of DCF can be monitored by fluorescence spectroscopy (Ex(max): 502nm, Em(max): 523nm), or by absorbance spectroscopy at 500nm (ε=79,500M-1cm-1). Neither nitric oxide, superoxide nor hydrogen peroxide alone appear to oxidize DCDHF.
Product Specific Literature References
Peroxynitrite-mediated oxidation of dihydrorhodamine 123: N.W. Kooy, et al.; Free Radic. Biol. Med. 16, 149 (1994) Abstract
Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species: J.P. Crow; Nitric Oxide 1, 145 (1997) Abstract
Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate: S.L. Hempel, et al.; Free Radic. Biol. Med. 27, 146 (1999) Abstract
Detection of reactive nitrogen species using 2,7-dichlorodihydrofluorescein and dihydrorhodamine 123: H. Ischiropoulos, et al.; Meth. Enzymol. 301, 367 (1999) Abstract
Further Categories Containing This Product:
Peroxynitrite/Scavengers/Detection
 
 
ALX-610-021 Revised 04-Nov-05
Dihydrorhodamine 123
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SYNONYMS DHR
PRODUCT LINE Other Products
PRODUCT CATEGORY Dyes/Stains/Fluorescent Probes/Fluorescent Labels
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ALX-610-021-M005   5 mg 153.00 USD Add To Cart
ALX-610-021-M010   10 mg 272.00 USD Add To Cart
Product Specification
FORMULA: C21H18N2O3
MW: 346.4
CAS NUMBER: 109244-58-8
PURITY: ≥98%
APPEARANCE: Pink crystalline solid.
SOLUBILITY: Soluble in organic solvents such as ethanol, DMSO or dimethyl formamide.
SHIPPING: AMBIENT
LONG TERM STORAGE: -20°C
USE/STABILITY: Stock solutions are stable for at least 6 months when stored at -20°C.

Product Description
Sensitive indicator of peroxynitrite formation. DHR is oxidized by peroxynitrite to the highly fluorescent product rhodamine. Formation of rhodamine can be monitored by fluorescence spectroscopy (Ex(max): 500nm, Em(max): 536nm), or by absorbance spectroscopy at 500nm (ε=78'800M-1cm-1). Neither nitric oxide, superoxide, nor hydrogen peroxide alone appear to oxidize DHR.
Product Specific Literature References
Peroxynitrite-mediated oxidation of dihydrorhodamine 123: N.W. Kooy, et al.; Free Radic. Biol. Med. 16, 149 (1994) Abstract
Attenuation of oxidation and nitration reactions of peroxynitrite by selenomethionine, selenocystine and ebselen: K. Briviba, et al.; Biochem. J. 319, 13 (1996) Abstract
Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species: J.P. Crow; Nitric Oxide 1, 145 (1997) Abstract
Glutathione peroxidase protects against peroxynitrite-mediated oxidations. A new function for selenoproteins as peroxynitrite reductase: H. Sies, et al.; J. Biol. Chem. 272, 27812 (1997) Abstract; Full Text
Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate: S.L. Hempel, et al.; Free Radic. Biol. Med. 27, 146 (1999) Abstract
Detection of reactive nitrogen species using 2,7-dichlorodihydrofluorescein and dihydrorhodamine 123: H. Ischiropoulos, et al.; Meth. Enzymol. 301, 367 (1999) Abstract
Further Categories Containing This Product:
Peroxynitrite/Scavengers/Detection • Respiratory Chain Other Products
 
 
ALX-270-097 Revised 07-Sep-06
Ebselen
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SYNONYMS 2-Phenyl-1,2-benzisoselenazol-3-(2H)-one
PRODUCT LINE Oxidative Stress
PRODUCT CATEGORY Peroxynitrite/Scavengers/Detection
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ALX-270-097-M001   1 mg 12.00 USD Add To Cart
ALX-270-097-M005   5 mg 20.00 USD Add To Cart
ALX-270-097-M025   25 mg 60.00 USD Add To Cart
Product Specification
FORMULA: C13H9NOSe
MW: 274.2
CAS NUMBER: 60940-34-3
PURITY: ≥98% (NMR)
APPEARANCE: White to off-white crystalline solid.
SOLUBILITY: Soluble in DMSO.
SHIPPING: AMBIENT
LONG TERM STORAGE: -20°C
HANDLING: Protect from light.

Product Description
Selenium-based inhibitor of protein kinase C, NADPH, 5-lipoxygenase, cyclooxygenase (COX) and NADPH oxidase. Anti-inflammatory antioxidant. Mimics glutathione peroxidase. Inhibits oxidative modifications of low density lipoproteins (LDL).
Product Specific Literature References
A novel biologically active seleno-organic compound--III. Effects of PZ 51 (Ebselen) on glutathione peroxidase and secretory activities of mouse macrophages: M.J. Parnham & S. Kindt; Biochem. Pharmacol. 33, 3247 (1984) Abstract
Seleno-organic compounds and the therapy of hydroperoxide-linked pathological conditions: M.J. Parnham & E. Graf; Biochem. Pharmacol. 36, 3095 (1987) Abstract
Kinetic mechanism and substrate specificity of glutathione peroxidase activity of ebselen (PZ51): M. Maiorino, et al.; Biochem. Pharmacol. 37, 2267 (1988) Abstract
Studies on the anti-inflammatory activity of ebselen. Ebselen interferes with granulocyte oxidative burst by dual inhibition of NADPH oxidase and protein kinase C?: I.A. Cotgreave, et al.; Biochem. Pharmacol. 38, 649 (1989) Abstract
T. Nikawa, et al.; Biochem. Pharmacol. 47, 1007 (1994) Abstract
Effects of ebselen and probucol on oxidative modifications of lipid and protein of low density lipoprotein induced by free radicals: N. Noguchi, et al.; Biochim. Biophys. Acta 1213, 176 (1994) Abstract
Strong inhibition of mammalian lipoxygenases by the antiinflammatory seleno-organic compound ebselen in the absence of glutathione: C. Schewe, et al.; Biochem. Pharmacol. 48, 65 (1994) Abstract
Ebselen: a glutathione peroxidase mimic: H. Sies; Meth. Enzymol. 234, 476 (1994) Abstract
Molecular actions of ebselen-an antiinflammatory antioxidant: T. Schewe; Gen. Pharmacol. 26, 1153 (1995) Abstract
Ebselen: H. Sies; Meth. Enzymol. 252, 341 (1995) Abstract
Ebselen as a glutathione peroxidase mimic and as a scavenger of peroxynitrite: H. Sies & H. Masumoto; Adv. Pharmacol. 38, 229 (1997) Abstract
Free radical generation by selenium compounds and their prooxidant toxicity: J.E. Spallholz; Biomed. Environ. Sci. 10, 260 (1997) Abstract
Protection against peroxynitrite by selenoproteins: H. Sies, et al.; Z. Naturforsch. [C] 53, 228 (1998) Abstract
Ebselen protects mice against T cell-dependent, TNF-mediated apoptotic liver injury: G. Tiegs, et al.; J. Pharmacol. Exp. Ther. 287, 1098 (1998) Abstract; Full Text
Further Categories Containing This Product:
PKC Inhibitors • Antioxidants, Flavonoids & Free Radical Scavengers Other Products • Malaria/Related Products • Lipoxygenases/Related Products • Anti-inflammatory Agents Other Products • COX Inhibitors
 
 
ALX-430-092 Revised 28-Jan-05
FeTMPyP . pentachloride
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SYNONYMS Iron (III) tetrakis(N-methyl-4'-pyridyl)porphyrin . 5Cl
PRODUCT LINE Oxidative Stress
PRODUCT CATEGORY Porphyrins
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ALX-430-092-M010   10 mg 43.00 USD Add To Cart
Product Specification
FORMULA: C44H36FeN8 . 5Cl
MW: 732.7 . 177.3
CAS NUMBER: 133314-07-5
PURITY: ≥98%
APPEARANCE: Black solid.
SOLUBILITY: Soluble in aqueous buffers or isotonic saline.
SHIPPING: SHIPPED ON BLUE ICE
LONG TERM STORAGE: -20°C
USE/STABILITY: Stable for at least 2 years after receipt when stored at –20°C.
HANDLING: Protect from light and moisture.

Product Description
Synthetic porphyrin complexed with iron which acts as a peroxynitrite decomposition complex. Catalyzes the isomerization of peroxynitrite to nitrate under physiological conditions. Effectively prevents the oxidation of lipid components by peroxynitrite. Also catalytically decomposes superoxide radicals. Irridation of the aqueous solution at 335nm releases hydroxyl radicals. It can also serve as a potential drug for reactive oxygen species (ROS) detoxification.
Product Specific Literature References
Peroxynitrite decomposition catalysts: M.K. Stern, et al.; JACS 118, 8735 (1996)
Amphiphilic peroxynitrite decomposition catalysts in liposomal assemblies: J.A. Hunt, et al.; Chem. Biol. 4, 845 (1997) Abstract
Peroxynitrite rapidly permeates phospholipid membranes: S.S. Marla, et al.; PNAS 94, 14243 (1997) Abstract
Characterization of the cytoprotective action of peroxynitrite decomposition catalysts: T.P. Misko, et al.; J. Biol. Chem. 273, 15646 (1998) Abstract; Full Text
Protective effects of a superoxide dismutase mimetic and peroxynitrite decomposition catalysts in endotoxin-induced intestinal damage: D. Salvemini, et al.; Br. J. Pharmacol. 127, 685 (1999) Abstract
Methamphetamine generates peroxynitrite and produces dopaminergic neurotoxicity in mice: protective effects of peroxynitrite decomposition catalyst: S.Z. Imam, et al.; Brain Res. 837, 15 (1999) Abstract
Further Categories Containing This Product:
Peroxynitrite/Scavengers/Detection
 
 
ALX-620-074 Revised 24-Jan-08
HPF
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SYNONYMS Hydroxyphenyl fluorescein
2-[6-(4'-Hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid
PRODUCT LINE Oxidative Stress
PRODUCT CATEGORY Oxidative Stress Markers & Reagents/Related Products
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Product Numbers: Format: Size: Unit Price: Quantity: Add To Cart
ALX-620-074-M001   1 mg 430.00 USD Add To Cart
Product Specification
FORMULA: C26H16O6
MW: 424.4
CONCENTRATION: ~5mM
PURITY: ≥98% (HPLC)
FORMULATION: Liquid. In 0.47ml dimethylformamide.
SHIPPING: AMBIENT
LONG TERM STORAGE: +4°C
USE/STABILITY:

Prepare 500-5'000-fold dilution (~10-1µM) in phosphate buffer (0.1M phosphate, pH 7.4) immediately before use. BSA, phenol red and amines may affect the fluorescence and must be used with caution. Do not store the dilutions.
HANDLING: Protect from light. Keep under inert gas. After opening, prepare aliquots and store at +4°C.
HAZARD: HARMFUL.

Product Images
Please click on thumbnails to enlarge.
Product Description
Cell permeable fluorescent reagent (Ex(max): 490nm; Em(max): 515nm) for the detection of highly reactive oxygen species (hROS). Immediately reacts with hROS such as hydroxyl radical and peroxynitrite, and the fluorescence intensity greatly increases. In addition, peroxynitrite can be detected in distinction from nitric oxide and superoxide since HPF does not react with nitric oxide, superoxide and hydrogen peroxide. Moreover, HPF is resistant to light-induced autooxidation. HPF does not react with hypochlorite (-OCl) either and thus can be used in combination with APF (Prod. No. ALX-620-075) which detects -OCl to elucidate reliably the roles of -OCl in biological systems such as neutrophils. Moreover, HPF is resistant to light-induced autooxidation. Not for sale in Japan.
Product Specific Literature References
Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species: K. Setsukinai, et al.; J. Biol. Chem. 278, 3170 (2003) Abstract
General Information
BACKGROUND/TECHNICAL INFORMATION Precautions for use:
1. Prepare the diluted solution just before use and use it up.
2. The dilution buffer should be adjusted to pH 7.0-7.5. Please note that bovine serum albumin (BSA) and phenol red might interfere with the fluorescence measurement.

Light-induced autoxidation (Figure 3)
After adding HPF or DCFH-DA (10µM), the cells were incubated for 30 min at 37°C in the dark. The fluorescence images were obtained with a confocal fluorescent microscope (excitation wavelength: 488nm, fluorescence emission wavelength: using a 515nm barrier filter). The cells were laser-irradiated again at 488nm for 10 sec and fluorescence images were obtained again (see Figure 3).

Fluorescence images of HPF- or APF-loaded neutrophils (Figure 4)
Neutrophils were obtained from porcine blood and suspended in Krebs-Ringer phosphate buffer (114mM NaCI, 4.6mM KCI, 2.4mM MgSO4, 1.0mM CaCI2, 15mM NaH2PO4/Na2HPO4, pH 7.4) and kept on ice until use. Then they were seeded onto a glass-bottomed dish. The cells were loaded with HPF or APF (10µM) by incubation for 30min at room temperature and then stimulated with PMA (including 2ng/ml; 0.1% DMF as a cosolvent). Fluorescence images were obtained with a confocal fluorescent microscope before and 10 min after the stimulation with PMA (excitation wavelength: 488nm, fluorescence emission wavelength: 505-550nm; using a 505-550nm barrier filter).

Detection of hROS in the HRP/H2O2 system using HPF and APF (Figure 5)
Reaction timecourse
Fluorescence probe reagents (final 10µM; 0.1% DMF as a cosolvent) were added to sodium phosphate buffer (0.1M; pH 7.4) containing HRP (0.2µM). H2O2 (final 1µM) was added at the time indicated by the arrow. The fluorescence intensities were measured at excitation wavelength of 490nm and fluorescence emission wavelength of 515nm. Relation between the amount of added H2O2 and fluorescence increase in the HRP/H2O2 system.

Relation between the amount of added H2O2 and fluorescence increase in the HRP/H2O2 system
Fluorescence probe reagents (final 10µM; 0.1% DMF as a cosolvent) were added to sodium phosphate buffer (0.1M; pH 7.4) containing HRP (0.2µM). The fluorescence intensities were measured at excitation wavelength of 490nm and fluorescence emission wavelength of 515nm.
MANUFACTURER Manufactured by Daiichi Pure Chemicals Co. Ltd., Japan.
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