Climate Change Data Portal
DOI | 10.3791/56945 |
Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-Encoded Fluorogenic Sensors | |
Corteselli, Elizabeth M.1; Samet, James M.2; Gibbs-Flournoy, Eugene3 | |
发表日期 | 2018-02-01 |
ISSN | 1940-087X |
期号 | 132 |
英文摘要 | 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 geneticallyencoded 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 (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, livecell 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 EGSH and H2O2 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. |
英文关键词 | Oxidative stress;Toxicology;Fluorescence;Live-cell imaging;Glutathione;Hydrogen peroxide |
语种 | 英语 |
WOS记录号 | WOS:000426453400058 |
来源期刊 | JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
![]() |
来源机构 | 美国环保署 |
文献类型 | 期刊论文 |
条目标识符 | http://gcip.llas.ac.cn/handle/2XKMVOVA/60386 |
作者单位 | 1.Univ North Carolina Chapel Hill, Dept Environm Sci & Engn, Gillings Sch Global Publ Hlth, Chapel Hill, NC USA; 2.Natl Hlth & Environm Effects Res Lab, Environm Publ Hlth Div, Chapel Hill, NC USA; 3.Oak Ridge Inst Sci & Educ, Oak Ridge, TN USA |
推荐引用方式 GB/T 7714 | Corteselli, Elizabeth M.,Samet, James M.,Gibbs-Flournoy, Eugene. Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-Encoded Fluorogenic Sensors[J]. 美国环保署,2018(132). |
APA | Corteselli, Elizabeth M.,Samet, James M.,&Gibbs-Flournoy, Eugene.(2018).Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-Encoded Fluorogenic Sensors.JOVE-JOURNAL OF VISUALIZED EXPERIMENTS(132). |
MLA | Corteselli, Elizabeth M.,et al."Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-Encoded Fluorogenic Sensors".JOVE-JOURNAL OF VISUALIZED EXPERIMENTS .132(2018). |
条目包含的文件 | 条目无相关文件。 |
除非特别说明,本系统中所有内容都受版权保护,并保留所有权利。