Integrated High-Content Quantification of Intracellular ROS Levels and Mitochondrial Morphofunction
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.
KeywordsReactive Oxygen Species Reactive Oxygen Species Level Intracellular Reactive Oxygen Species Reactive Nitrogen Species Increase Reactive Oxygen Species Production
This research was supported by the University of Antwerp (TTBOF/UA_29267), the Special Research Fund of Ghent University (project BOF/11267/09), the Hercules Foundation (AUGE/ 013), NB-Photonics (Project code 01-MR0110), and the CSBR (Centers for Systems Biology Research) initiative from the Netherlands Organization for Scientific Research (NWO; No: CSBR09/013V).
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