Fluorogenic probes applied to the study of induced oxidative stress in thehuman leukemic HL60 cell line

Development of microspectrofluorometric methods using specific fluorogenic probes has provided precious help in studying in situ oxidative stress and cellular protective systems. The aim of this study was to determine ROS pro duction concomitantly with a modification of the intracellular thiol pool a fter applying an oxidative stress to a nonadherent cell model represented b y the HL60 cell line. The dichlorodihydrofluorescein diacetate (H(2)DCFDA) probe assessed the kinetic production of ROS by cells submitted to the chem ical oxidant t-butylhydroperoxide with a high signal/noise ratio. The probe sensitivity permitted us to detect endogenous ROS production in HL60 cells and the protective effect of N-acetyl cysteine against ROS. The chlorometh ylfluorescein diacetate probe (CMFDA) permitted us to evaluate the thiol de pleting effect of N-ethyl maleimide. Complete thiol depletion was associate d with a moderate increase in ROS production. The cell viability was determ ined with calcein-AM, which gave results similar to those with the tetrazol ium dye. This probe was not affected by intracellular pH and did not requir ed an extraction step, unlike tetrazolium dye. In conclusion, cell-permeant fluorogenic probes are useful and sensitive tools to determine in situ ROS production concomitantly with consecutive change in the thiol system in a living and nonadherent cell model.