title: Integrated High-Content Quantification of Intracellular ROS Levels and Mitochondrial Morphofunction.
abstract: Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-content microscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells.
authors.affiliation: Cell Systems and Imaging Research Group (CSI), Department of Molecular Biotechnology, Ghent University, Ghent, Belgium.|Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: Tom
authors.initials: T
authors.lastname: Sieprath
authors.affiliation: Cell Systems and Imaging Research Group (CSI), Department of Molecular Biotechnology, Ghent University, Ghent, Belgium.|Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: Tobias D J
authors.initials: TD
authors.lastname: Corne
authors.affiliation: Department of Biochemistry (286), Radboud University Medical Centre (RUMC), Radboud Institute for Molecular Life Sciences (RIMLS), Nijmegen, The Netherlands.
authors.forename: Peter H G M
authors.initials: PH
authors.lastname: Willems
authors.affiliation: Department of Biochemistry (286), Radboud University Medical Centre (RUMC), Radboud Institute for Molecular Life Sciences (RIMLS), Nijmegen, The Netherlands.
authors.forename: Werner J H
authors.initials: WJ
authors.lastname: Koopman
authors.affiliation: Cell Systems and Imaging Research Group (CSI), Department of Molecular Biotechnology, Ghent University, Ghent, Belgium. winnok.devos@uantwerpen.be.|Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium. winnok.devos@uantwerpen.be.
authors.forename: Winnok H
authors.initials: WH
authors.lastname: De Vos
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D005057:Eukaryotic Cells / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods / Q000706:statistics & numerical data*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism*; D057188:Workflow
chemical_list: D000975:Antioxidants; D017382:Reactive Oxygen Species