Monitoring Changes in the Oxidizing Milieu in the Endoplasmic Reticulum of Mammalian Cells Using HyPerER

Julia Birk; Beata Lizak; Christian Appenzeller-Herzog; Alex Odermatt

doi:10.21769/BioProtoc.4076

Monitoring Changes in the Oxidizing Milieu in the Endoplasmic Reticulum of Mammalian Cells Using HyPerER

Bio Protoc. 2021 Jul 5;11(13):e4076. doi: 10.21769/BioProtoc.4076.

Authors

Julia Birk¹, Beata Lizak², Christian Appenzeller-Herzog^{1

3}, Alex Odermatt¹

Affiliations

¹ Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland.
² Department of Molecular Biology, Semmelweis University, Budapest, Hungary.
³ University Medical Library, University of Basel, Basel, Switzerland.

Abstract

The production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress are tightly linked. The generation of ROS can be both the cause and a consequence of ER stress pathways, and an increasing number of human diseases are characterized by tissue atrophy in response to ER stress and oxidative injury. For the assessment of modulators of ER luminal ROS generation and for mechanistic studies, methods to monitor changes in ER reduction-oxidation (redox) states in a time-resolved and organelle-specific manner are needed. This has been greatly facilitated by the development of genetically encoded fluorescent probes, which can be targeted to different subcellular locations by specific amino acid extensions. One of these probes is the yellow fluorescent protein-based redox biosensor, HyPer. Here, we provide a protocol for the time-resolved monitoring of the oxidizing milieu in the ER of adherent mammalian cells using the ratiometric sensor, HyPerER, which is specifically targeted to the ER lumen.

Keywords: Endoplasmic reticulum; Fluorescence microscopy; HyPer; Hydrogen peroxide; Oxidative stress; Redox.