Photobleaching of fluorescein as a probe for oxidative stress in single cells.

Background: ROS are involved in the regulation of many physiological and pathological processes. Apoptosis and necrosis are processes that are induced by changes in concentrations of Reactive Oxygen Species (ROS). This study aims to detect and quantify the cellular response to changing ROS concentrations in the scope of apoptosis and necrosis.

Methods: Photobleaching of the fluorescent substrate fluorescein is used as a probe to detect the response of individual Jurkat-T-lymphocytes and Prostate-Cancer-3(PC-3) cells to oxidative stress, induced by hydrogen peroxide (H₂O₂). A kinetic model is proposed to describe changes in intracellular dye quantities due to photobleaching, dye hydrolysis, influx and leakage, yielding a single time-dependent decaying exponent+constant.

Results: Fluorescein photobleaching is controlled and used to detect intracellular ROS. An increase in the decay time of fluorescence of intracellular fluorescein (slow photobleaching) was measured from cells incubated with H₂O₂ at 50 μM. At higher H₂O₂ concentrations a decrease in the decay time was measured (fast photobleaching), in contrast to in vitro results with fluorescein and H₂O₂ in phosphate buffer saline (PBS), where the addition of H₂O₂ decreases the decay time, regardless of the irradiation dose used.

Conclusions: The anomalous, ROS-concentration dependent reduction of the photobleaching rate in cells, as opposed to solutions, might indicate on the regulation of the activity of intracellular oxidative-stress protective mechanisms, as seen earlier with other methods.

Significance: Assessing photobleaching via the time decay of the fluorescence intensity of an ROS-sensitive fluorophore may be adapted to monitor oxidative stress or ROS-related processes in cells.