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-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 (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.