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

Max Platkov, Reuven Tirosh, Menahem Kaufman, Naomi Zurgil, Mordechai Deutsch

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

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 H2O2at 50 μM. At higher H2O2concentrations a decrease in the decay time was measured (fast photobleaching), in contrast to in vitro results with fluorescein and H2O2in phosphate buffer saline (PBS), where the addition of H2O2decreases 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.

Original languageEnglish
Pages (from-to)306-314
Number of pages9
JournalJournal of Photochemistry and Photobiology B: Biology
Volume140
DOIs
StatePublished - Nov 2014

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© 2014 Elsevier B.V. All rights reserved.

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