Optimization of experimental and computational processes to study mitochondrial trafficking and metabolic biomarkers
Summary, in English
Material and methods: The neuroblastoma cell line SH-SY5Y was used and differentiated using different protocols to develop a neuronal phenotype. To disturb mitochondrial metabolism and traffic, two mitochondrial poisons, rotenone and 6-hydroxydopamine, were used. Cells were labelled with different fluorescent probes and imaged under a INCell Analyzer. Cell mass and metabolic activity were measured using the SRB and resazurin assays, respectively.
Results: We have shown that a seven-day cell differentiation protocol using retinoic acid provided cells with better neuronal-like morphology, which are suited to study mitochondrial metabolic and dynamic parameters. We also demonstrate that rotenone and 6-hydroxydopamine toxicity is time and dose dependent, altering cell and mitochondria morphology, as well as mitochondrial membrane potential.
Conclusions: Images obtained with increasing concentrations of mitochondrial toxicants showed a gradual effect on different mitochondrial parameters that can be quantified using computational tools. These effects will be paired with metabolic data obtained for the same drug concentrations at later timepoints, to assess the consequences of mitochondrial disruption on cell viability. Building on the collected experimental information, we are training machine learning classification algorithms to accurately predict point-of-no-return situations that unbalance the cell to a state of metabolic catastrophe.
52nd Annual Meeting of the European Society for Clinical Investigation
2018-05-30 - 2018-06-01
- ISSN: 0014-2972