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

Paper ID Volume ID Publish Year Pages File Format Full-Text
29958 44448 2014 9 PDF Available
Title
Photobleaching of fluorescein as a probe for oxidative stress in single cells
Abstract

•Fluorescein photobleaching is controlled and used to detect intracellular ROS.•Unexpectedly, low ROS concentration slows photobleaching in cells.•Higher ROS concentrations accelerate photobleaching in cells.•A kinetic model describes intracellular dye phases by a single exponent + constant.

BackgroundROS 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.MethodsPhotobleaching 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.ResultsFluorescein 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.ConclusionsThe 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.SignificanceAssessing 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.

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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Photochemistry and Photobiology B: Biology - Volume 140, November 2014, Pages 306–314
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us