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IMAGING APPROACHES TO ASSESSMENTS OF TOXICOLOGICAL OXIDATIVE STRESS USING GENETICALLY ENCODED FLUOROGENIC SENSORS
Citation:
Corteselli, E., J. Samet, AND E. Gibbs-Flournoy. IMAGING APPROACHES TO ASSESSMENTS OF TOXICOLOGICAL OXIDATIVE STRESS USING GENETICALLY ENCODED FLUOROGENIC SENSORS. Journal of Visualized Experiments . JoVE, Somerville, MA, 132:56945, (2018). https://doi.org/10.3791/56945
Impact/Purpose:
This is an invited methods review article on recent advances in the use of imaging techniques for the analysis of oxidative cellular stress induced by toxicological exposures. The objective of the article is educational in that it is written as a practical guide for laboratory scientists seeking to make use of the high spatiotemporal resolution of live cell microscopy to study mechanisms of cellular injury involved in the toxicity of environmental oxidants.
Description:
Live-cell imaging using genetically encoded fluorogenic reporters of the intracellular glutathione redox potential and hydrogen peroxide is an experimental approach that offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress. While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H202). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors radiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H202 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in livecell toxicological assessments of oxidative stress.
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DOI: IMAGING APPROACHES TO ASSESSMENTS OF TOXICOLOGICAL OXIDATIVE STRESS USING GENETICALLY ENCODED FLUOROGENIC SENSORS