Fluorogenic Probes Applied to the Study of Induced Oxidative Stress in the Human Leukemic HL60 Cell Line

Abstract

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 production concomitantly with a modification of the intracellular thiol pool after applying an oxidative stress to a nonadherent cell model represented by the HL60 cell line. The dichlorodihydrofluorescein diacetate (H₂DCFDA) probe assessed the kinetic production of ROS by cells submitted to the chemical 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 chloromethylfluorescein diacetate probe (CMFDA) permitted us to evaluate the thiol depleting effect of N-ethyl maleimide. Complete thiol depletion was associated with a moderate increase in ROS production. The cell viability was determined with calcein-AM, which gave results similar to those with the tetrazolium dye. This probe was not affected by intracellular pH and did not required 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 non-adherent cell model.