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Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells
Author(s) -
Wayne O. Carter,
Padma K. Narayanan,
J. Paul Robinson
Publication year - 1994
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1002/jlb.55.2.253
Subject(s) - dichlorofluorescein , superoxide , endothelial stem cell , intracellular , xanthine oxidase , superoxide dismutase , biochemistry , reactive oxygen species , endothelium , biology , microbiology and biotechnology , hydrogen peroxide , chemistry , oxidative stress , in vitro , endocrinology , enzyme
One of the objectives of studying endothelial cells in vitro is to evaluate neutrophil‐endothelial cell interactions including potential consequences of oxidant‐mediated damage to the endothelial cell. Current under‐standing of endothelial cell oxidative function is derived primarily from the measurement of extracellular products. We utilized 2 dyes, 2′,7′‐dichlorofluorescin diacetate (DGFH‐DA) and hydroethidine (HE), which measure hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2 ‐ ) respectively, for their suitability to monitor oxidative mechanisms in endothelial cells and to provide a reliable measure of intracellular oxidants. Endothelial cells stained with DCFH‐DA and stimulated with H 2 O 2 exhibited an increase in the fluorescent product 2′,7′‐dichlorofluorescein (DCF) (measure of intracellular H 2 O 2 ) which peaked at 10 min. Endothelial cells stained with HE and stimulated with H 2 O 2 exhibited an increase in the fluorescent product ethidium bromide (EB) (measure of intracellular O 2 ‐ ) which lasted for approximately 60 min. Superoxide dismutase increased DCF fluorescence in endothelial cells stimulated with H 2 O 2 by 158%. Allopurinol (xanthine oxidase inhibitor) reduced DCF and EB fluorescence by 48% and 37% respectively in endothelial cells stimulated with H 2 O 2 . Catalase completely inhibited an increase in DCF or EB fluorescence in endothelial cells stimulated with H 2 O 2 . There was a direct correlation between mean DCF and EB fluorescence intensity and the concentration of H 2 O 2 or the number of phorbol 12‐myristate 13‐acetate‐activated neutrophils added to endothelial cells. We conclude from these studies that DCFH‐DA and HE can be used to measure intracellular H 2 O 2 and O 2 ‐ in endothelial cells and that the xanthine oxidase pathway for intracellular O 2 ‐ production accounts for approximately 40% of the total intracellular O 2 ‐ generated in endothelial cells after stimulation with H 2 O 2 . The combination of image cytometry and flow cytometry will be important for future evaluations of endothelial cell function. J. Leukoc. Biol. 55: 253–258; 1994.