APF |
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| SYNONYMS | Aminophenyl fluorescein
2-[6-(4'-Amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid |
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| PRODUCT LINE | Oxidative Stress |
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| PRODUCT FAMILIES | Oxidative Stress Markers & Reagents / Related Products |
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| Ordering Information |
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| Product Numbers | Format | Size | Unit Price | Quantity | | | ALX-620-075-M001 |
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1 mg | | | | |  | |
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| Product Specification |
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| FORMULA: | C26H17NO5 |
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| MW: | 423.4 |
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| CAS NUMBER: | 359010-70-1 |
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| CONCENTRATION: | ~5mM |
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| PURITY: | ≥98% (HPLC) |
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| FORMULATION: | Liquid. In 0.47ml dimethyl formamide. |
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| SHIPPING: | AMBIENT |
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| LONG TERM STORAGE: | +4蚓 |
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| USE/STABILITY: | Prepare 500-5’000-fold dilution (~10-1然) 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. |
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| HANDLING: | Protect from light. Keep under inert gas. After opening, prepare aliquots and store at +4蚓. |
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| 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. |
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| | 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 |
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| | 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 (10然), the cells were incubated for 30 min at 37蚓 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 (10然) 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 10然; 0.1% DMF as a cosolvent) were added to sodium phosphate buffer (0.1M; pH 7.4) containing HRP (0.2然). H2O2 (final 1然) 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 10然; 0.1% DMF as a cosolvent) were added to sodium phosphate buffer (0.1M; pH 7.4) containing HRP (0.2然). The fluorescence intensities were measured at excitation wavelength of 490nm and fluorescence emission wavelength of 515nm. | | |
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| Related Products |
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| | HPF | |
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| RELATED PRODUCT GROUPS |
Oxidative Stress > Peroxynitrite / Scavengers / Detection / Other Products > Dyes / Stains / Fluorescent Probes |
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