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 multi-parametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells.
The above study was carried out within the RIMLS and Radboud Center for Mitochondrial Medicine (RCMM) in a collaborative effort between the Department of Biochemistry (286) of the Radboudumc, the Cell Systems and Imaging Group (Ghent University Belgium) and the Laboratory of Cell Biology and Histology (University of Antwerp, Belgium).
The paper was published as: T. Sieprath, T.D. Corne, P.H.G.M Willems, Werner J.H. Koopman and W.H. De Vos. Integrated high-content quantification of intracellular ROS levels and mitochondrial morphofunction (2016) Adv. Anat. Embryol. Cell Biol. 219:149-177.
Impact factor: 17
Average Impact factor (5 years): 6.92
Category: Anatomy and Morphology
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