ALEXIS Biochemicals: HPF

ALX-620-074

Revised 24-Jan-08

HPF
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

Ordering Information

Product Numbers:	Format:	Size:	Unit Price:	Quantity:	Add To Cart
ALX-620-074-M001		1 mg	430.00 USD

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/H₂O₂system 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.