High-content analysis of cellular oxidative stress

Reactive oxygen species (ROS) are small, short lived molecules that mediate various cellular responses including cell proliferation, differentiation, gene expression and migration. Excessive accumulation of ROS however, can lead to DNA damage and the build-up of irreversibly oxidized proteins. To counter the potential damaging effects of ROS, cells have evolved several antioxidant systems, including ROS defusing enzymes and vitamins. An imbalance between production of oxidative radicals and antioxidant mechanisms induces a state of oxidative stress, a phenomenon associated with various systemic diseases and aging. One of the major sources of ROS is the mitochondrion, which harbours the electron transport chain, handling highly active electrons in close proximity to oxygen. To study the relationship between ROS levels and mitochondrial (dys-) function in various contexts, we have developed an integrated workflow for measurement of intracellular ROS levels, mitochondrial potential and mitochondrial morphology. To this end, human dermal fibroblasts, cultured in glass-bottom 96-well plates, were stained with a fluorescent ROS-sensitive dye (pan-cellular (CM-H2DCFDA) or mitochondrially targeted (MitoSOX)), or a mitochondrial membrane potential-sensitive dye (TMRM), after which they were acquired in a fully automated manner and analysed using home-written image analysis protocols (RedoxMetrics and MitoMetrics). In fluxo chemical perturbation allowed determining the dynamic range and sensitivity towards the applied stressors. In case of mitochondrial analysis, mitochondria were specifically enhanced using a multiscale Laplacian operator and an exhaustive morphological and textural feature extraction was performed. Using this approach, we showed that specific chemical perturbations selectively affect cellular ROS levels or mitochondrial morphology.

Publication year:2014