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Updated: Jul 19, 2026
Quantification of Reactive Oxygen Species Using 2′,7′-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells
Published on: May 13, 2022
Simultaneous quantification of oxidative stress and cell spreading using
Werner J H Koopman1, Sjoerd Verkaart, Sjenet E van Emst-de Vries
1Department of Membrane Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
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.
