Measuring Mitochondrial ROS in Mammalian Cells with a Genetically Encoded Protein Sensor

Xin Zhang; Christine Silvia Gibhardt; Sabrina Cappello; Katharina Maria Zimmermann; Adina Vultur; Ivan Bogeski

doi:10.21769/BioProtoc.2705

Measuring Mitochondrial ROS in Mammalian Cells with a Genetically Encoded Protein Sensor

Bio Protoc. 2018 Jan 20;8(2):e2705. doi: 10.21769/BioProtoc.2705.

Authors

Xin Zhang^{1

1}, Christine Silvia Gibhardt¹, Sabrina Cappello¹, Katharina Maria Zimmermann², Adina Vultur¹, Ivan Bogeski¹

Affiliations

¹ Molecular Physiology, Institute of Cardiovascular Physiology, University Medical Center, University of Göttingen, Göttingen, Germany.
² Department of Biophysics, CIPMM, School of Medicine, Saarland University, 66421 Homburg, Germany.

Abstract

Reactive oxygen species (ROS) are not only known for their toxic effects on cells, but they also play an important role as second messengers. As such, they control a variety of cellular functions such as proliferation, metabolism, differentiation and apoptosis. Thus, ROS are involved in the regulation of multiple physiological and pathophysiological processes. It is now apparent that there are transient and local changes in ROS in the cell; in so-called 'microdomains' or in specific cellular compartments, which affect signaling events. These ROS hotspots need to be studied in more depth to understand their function and regulation. Therefore, it is necessary to identify and quantify redox signals in single cells with high spatial and temporal resolution. Genetically encoded fluorescence-based protein sensors provide such necessary tools to examine redox-signaling processes. A big advantage of these sensors is the possibility to target them specifically. Mitochondria are essential for energy metabolism and are one of the major sources of ROS in mammalian cells. Therefore, the evaluation of redox potential and ROS production in these organelles is of great interest. Herein, we provide a protocol for the real-time visualization of mitochondrial hydrogen peroxide (H₂O₂) using the H₂O₂-specific ratiometric sensor mitoHyPer in adherent mammalian cells.

Keywords: Fluorescence microscopy; HyPer; Mammalian cell; Mitochondrial ROS; Protein sensor; Real-time imaging; roGFP-Orp1.