Simultaneous quantification of oxidative stress and cell spreading using 5‐(and‐6)‐chloromethyl‐2′,7′‐dichlorofluorescein

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Abstract
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‐H2DCF), 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‐H2DCF] was not rate‐limiting and that the rate of [CM‐DCF] formation (VCM‐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. © 2006 International Society for Analytical Cytology