1. Simultaneous quantification of oxidative stress and cell spreading using 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein
- Author
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Koopman, W.J.H., Verkaart, S.A.J., Emst-de Vries, S.E. van, Grefte, S., Smeitink, J.A.M., and Willems, P.H.G.M.
- Subjects
Mitochondrial medicine [IGMD 8] ,Energy and redox metabolism [NCMLS 4] ,Cellular energy metabolism [UMCN 5.3] ,Renal disorder [IGMD 9] - Abstract
Contains fulltext : 50403.pdf (Publisher’s version ) (Closed access) BACKGROUND: Mitochondrial dysfunction may lead to increased oxidative stress and consequent changes in cell spreading. Here, we describe and validate a novel method for simultaneous quantification of these two parameters. METHODS: Human skin fibroblasts were loaded with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-H(2)DCF), and its oxidative conversion into CM-DCF was monitored as a function of time by video-rate confocal microscopy and real-time image averaging. Cell size was determined after binarization of the acquired images. RESULTS: At the lowest practical laser output, CM-DCF formation occurred with zero order kinetics, indicating that [CM-H(2)DCF] was not rate-limiting and that the rate of [CM-DCF] formation (V(CM-DCF)) was a function of the cellular oxidant level. Analysis of fibroblasts of a healthy control subject and a patient with a deficiency of NADH:ubiquinone oxidoreductase, the first complex of the oxidative phosphorylation system, revealed a significant increase in cellular oxidant level in the latter cells that was, however, not accompanied by a change in cell spreading. Conversely, chronic treatment with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), a derivative of vitamin E, markedly decreased the oxidant level and cell spreading in both control and patient fibroblasts. CONCLUSIONS: We present a reliable method for simultaneous quantification of oxidant levels and cell spreading in living cells.
- Published
- 2006