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Cellular redox profiling using high-content microscopy

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Abstract
Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (Delta psi(m)) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H(2)DCFDA (ROS) and TMRM (Delta psi(m) and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.
Keywords
Cellular Biology, Issue 123, Reactive Oxygen Species, Mitochondria, Mitochondrial Morphofunction, Live Cell Imaging, High-content Microscopy, Fluorescence Microscopy, Quantitative Multiparametric Microscopy, Redox Biology, OXYGEN SPECIES PRODUCTION, HIV PROTEASE INHIBITOR, OXIDATIVE STRESS, MITOCHONDRIAL MORPHOFUNCTION, CELLS, DYNAMICS, HYPERPOLARIZATION, QUANTIFICATION, DEFICIENCY, APOPTOSIS

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Citation

Please use this url to cite or link to this publication:

Chicago
Sieprath, Tom, Tobias Corne, Joke Robijns, Werner JH Koopman, and Winnok De Vos. 2017. “Cellular Redox Profiling Using High-content Microscopy.” Jove-journal of Visualized Experiments (123).
APA
Sieprath, T., Corne, T., Robijns, J., Koopman, W. J., & De Vos, W. (2017). Cellular redox profiling using high-content microscopy. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, (123).
Vancouver
1.
Sieprath T, Corne T, Robijns J, Koopman WJ, De Vos W. Cellular redox profiling using high-content microscopy. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS. 2017;(123).
MLA
Sieprath, Tom, Tobias Corne, Joke Robijns, et al. “Cellular Redox Profiling Using High-content Microscopy.” JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 123 (2017): n. pag. Print.
@article{8525129,
  abstract     = {Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (Delta psi(m)) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H(2)DCFDA (ROS) and TMRM (Delta psi(m) and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.},
  articleno    = {e55449},
  author       = {Sieprath, Tom and Corne, Tobias and Robijns, Joke and Koopman, Werner JH and De Vos, Winnok},
  issn         = {1940-087X},
  journal      = {JOVE-JOURNAL OF VISUALIZED EXPERIMENTS},
  language     = {eng},
  number       = {123},
  pages        = {14},
  title        = {Cellular redox profiling using high-content microscopy},
  url          = {http://dx.doi.org/10.3791/55449},
  year         = {2017},
}

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