Hydrogen peroxide release from human eosinophils on fibronectin: scopoletin enhances eosinophil activation.
We have examined the release of H(2)O(2) from PAF or TNFalpha-stimulated human eosinophils on fibronectin (FN)-coated polystyrene plates. H(2)O(2) release was measured by the standard scopoletin-horseradish peroxidase (SCOP-HRP) method and compared with that measured by a new microplate fluorescent assay for H(2)O(2) using a novel HRP substrate A6550. We observed that the SCOP-HRP method gave a 25-fold higher estimate of H(2)O(2) release from eosinophils than did the A6550-HRP method. Microscopic examination of PAF or TNFalpha-stimulated eosinophils in buffer alone or A6550-HRP reaction mixture showed that the cells remained generally round, while eosinophils in SCOP-HRP reaction mixture were spread on the fibronectin-coated surface. Measurement of the cellular ATP content after PAF-stimulation showed that only eosinophils activated in SCOP-HRP had a 50% fall in ATP content. This supported our conclusion that measurement of H(2)O(2) release from eosinophils in SCOP-HRP reaction mixture is problematic since the SCOP-HRP system activates eosinophils. However, we also found that A6550-HRP, when present throughout the incubation, resulted in a lower estimate of H(2)O(2) release than expected. The method used to detect eosinophil H(2)O(2) release greatly influences the absolute amount of H(2)O(2) detected.[1]References
- Hydrogen peroxide release from human eosinophils on fibronectin: scopoletin enhances eosinophil activation. Raible, D.G., Mohanty, J.G., Jaffe, J.S., Stella, H.J., Sprenkle, B.E., Glaum, M.C., Schulman, E.S. Free Radic. Biol. Med. (2000) [Pubmed]
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