ANALYTICAL AND NUMERICAL TECHNIQUES FOR THE EVALUATION OF FREE-RADICAL DAMAGE IN CULTURED-CELLS USING SCANNING LASER MICROSCOPY

Scanning laser microscopy (SLM) was used to develop an assay to visual ize the generation of intracellular reactive oxygen species (ROS) and to evaluate the effect of the lipophilic antioxidant U-87,663 on ROS f ormation. Cultured N18 neuroglioma cells were challenged by extracellu lar addition of cumene hydroperoxide, and subsequent intracellular gen eration of ROS was characterized by measuring the fluorescence Intensi ty of the ROS indicator 2',7'-dichlorofluorescein (DCF), The kinetics of the reaction between ROS and the indicator DCF, or the antioxidant U-87,663, can be most accurately assessed if results from individual c ell clusters are analysed independently, It is possible and necessary to account for these experimental and analytical properties in order t o characterize the properties of the antioxidant activity precisely, W e determined that the temporal increase in DCF fluorescence was consis tent with the reaction of DCF with free radicals generated from cumene hydroperoxide, as was the loss of fluorescence from U-87,663. The rat e constants for the free radical reactions revealed that ROS reaction with DCF is 10 times faster than with U-87,663, These differences in r eaction rates combined with differences in the cellular distribution o f the ROS indicator DCF, the antioxidant U-87,663, and the built: of t he ROS prevented detection of any protection U-87,663 may offer. (C) 1 996 Wiley-liss, Inc.