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- Title
Use of 2′,7′-Dichlorodihydrofluorescein diacetate in an Easy and Quantifiable Assay for Hypochlorous Acid Oxidation.
- Authors
Wu, Tina T.; Kai Chen; Keaney, John F.
- Abstract
Hypochlorous acid (HOCl) is a potent 2-electron oxidant produced by myeloperoxidase (MPO) under inflammatory conditions and is capable of reacting with a wide array of biological molecules. Here, we describe a straightforward and quantifiable assay in a 96-well microtiter plate format that allows assessment of HOCl-induced oxidation using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) that is more sensitive, and allows a lower limit of detection than standard BSA protein-carbonyl formation assays. We found that HOCl (1 to 250µM) caused a dose-dependent increase in DCFH-DA fluorescence. Specifically, 1µM HOCl caused a 13-fold increase in DCFH-DA fluorescence and only a 1-fold increase in BSA carbonylation. Treatment with H202 and NO had no impact on the assay. Thiol-containing amino acids that demonstrated interference with the BSA carbonyl assay were successfully evaluated with our assay. Effects of these compounds on inhibiting HOCl-induced cell death was examined using Trypan blue exclusion dye. Finally, UVVIS spectroscopy indicated formation of new reaction product as a result of HOCl and DCFH-DA. Thus, our simple and rapid assay should prove useful in both cell culture studies and for testing compounds for potential reaction with HOCl, an oxidant of increasing interest with regard to vascular disease.
- Subjects
OXIDIZING agents; BIOMOLECULES; PHYSIOLOGICAL oxidation; THIOLS; AMINO acids; CELL death; CELL culture; VASCULAR diseases
- Publication
FASEB Journal, 2007, Vol 21, Issue 6, pA1172
- ISSN
0892-6638
- Publication type
Article
- DOI
10.1096/fasebj.21.6.a1172-d