Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors
| Autor: | James M. Samet, Elizabeth M. Corteselli, Eugene A. Gibbs-Flournoy |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Cancer Research General Chemical Engineering Biosensing Techniques medicine.disease_cause Fluorescence General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Confocal imaging Live cell imaging Microscopy medicine Image Processing Computer-Assisted Fluorescent Dyes Hydrogen peroxide metabolism General Immunology and Microbiology Chemistry General Neuroscience Oxidation reduction Hydrogen Peroxide Fluorescence intensity Oxidative Stress 030104 developmental biology Biological system Oxidation-Reduction Oxidative stress |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/56945-v |
| Popis: | 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 (E(GSH)), while HyPer directly detects hydrogen peroxide (H(2)O(2)). 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 ratiometric, 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 E(GSH) and H(2)O(2) 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 live-cell toxicological assessments of oxidative stress. |
| Databáze: | OpenAIRE |
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