Simultaneous quantification of oxidative stress and cell spreading using 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein.
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.
Koopman
Werner J H
WJ
Department of Membrane Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Verkaart
Sjoerd
S
van Emst-de Vries
Sjenet E
SE
Grefte
Sander
S
Smeitink
Jan A M
JA
Willems
Peter H G M
PH
D000975:Antioxidants / Q000494:pharmacology; D002465:Cell Movement; D048429:Cell Size; D005347:Fibroblasts / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry* / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000493:pharmacokinetics; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*; D018715:Microscopy, Video / Q000379:methods; D016877:Oxidants / Q000032:analysis*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D012867:Skin / Q000737:chemistry / Q000166:cytology
D000975:Antioxidants; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016877:Oxidants; C037631:2',7'-dichlorofluorescein