Abstract 512: Cell-based analysis of oxidative stress, lipid peroxidation and lipid peroxidation-derived protein modifications using fluorescence microscopy

Abstract

Abstract Oxidative stress plays an important role in the progression of several diseases including inflammation, atherosclerosis, aging and age-related degenerative disorders. Reactive oxygen species damage membrane bound lipids resulting in lipid peroxidation-derived protein modifications. Cell-based measurements of oxidative stress, lipid peroxidation and protein carbonylation by traditional fluorescence microscopy provide a powerful platform to quantitate oxidative stress and lipid peroxidation. Here, we used three different approaches to measure oxidative stress and lipid peroxidation in cells by fluorescence microscopy. 1) Two new fluorogenic probes, CellROX™ Orange and CellROX ™ Green Reagents to measure oxidative stress in cells, 2) Image-iT® Lipid Peroxidation Kit for a ratiometric determination of lipid peroxidation in live cells 3) Click-iT® Lipid Peroxidation Imaging Kit, a click chemistry-based approach which utilizes incorporation of an alkyne-modified unsaturated fatty acid analog, linoleamide, into the cellular membranes. The resulting oxidation products, like 9, 12-dioxo-10(E) dodecenoic acid (DODE) can readily modify proteins and these modifications were readily detected in fixed cells by the copper-catalyzed click reaction using fluorescent azides. Using these approaches, we measured oxidative stress and lipid peroxidation caused by several oxidants in cells. Increases in oxidative stress, lipid peroxidation, and protein modifications were assessed by high content imaging and analysis as well as traditional fluorescence microscopy. In the models tested, at least 2-3 fold increases were observed compared to controls was and responses were successfully inhibited by antioxidants. The three strategies described here provide powerful new tools for the assessment of oxidative stress in cells and convey distinct advantages over existing cell-based methods. Citation Format: Bhaskar S. Mandavilli, Robert Aggeler. Cell-based analysis of oxidative stress, lipid peroxidation and lipid peroxidation-derived protein modifications using fluorescence microscopy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 512. doi:10.1158/1538-7445.AM2014-512