Abstract
Cellular phenotyping of human dermal fibroblasts (HDFs) from patients with inherited diseases provides invaluable information for diagnosis, disease aetiology, prognosis and assessing of treatment options. Here we present a cell phenotyping protocol using image cytometry that combines measurements of crucial cellular and mitochondrial parameters: (1) cell number and viability, (2) thiol redox status (TRS), (3) mitochondrial membrane potential (MMP) and (4) mitochondrial superoxide levels (MSLs). With our protocol, cell viability, TRS and MMP can be measured in one small cell sample and MSL on a parallel one. We analysed HDFs from healthy individuals after treatment with various concentrations of hydrogen peroxide (H2O2) for different intervals, to mimic the physiological effects of oxidative stress. Our results show that cell number, viability, TRS and MMP decreased, while MSL increased both in a time- and concentration-dependent manner. To assess the use of our protocol for analysis of HDFs from patients with inherited diseases, we analysed HDFs from two patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD), one with a severe clinical phenotype and one with a mild one. HDFs from both patients displayed increased MSL without H2O2 treatment. Treatment with H2O2 revealed significant differences in MMP and MSL between HDFs from the mild and the severe patient. Our results establish the capacity of our protocol for fast analysis of cellular and mitochondrial parameters by image cytometry in HDFs from patients with inherited metabolic diseases.
Competing interests: None declared
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Acknowledgements
We acknowledge Christian Knudsen, Department of Biomedicine, Aarhus University, Aarhus, for technical assistance as well as the Department of Clinical Medicine and the Faculty of Health Sciences at Aarhus University, Aarhus, for financial support.
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Communicated by: Piero Rinaldo, MD, PhD
Take-Home Message
Protocol for cellular and mitochondrial phenotyping reveals differences between fibroblasts from VLCADD patients with mild and severe gene variations.
Compliance with Ethical Guidelines
Conflict of Interest
Paula Fernandez Guerra, Martin Lund, Thomas Juhl Corydon, Nanna Cornelius, Niels Gregersen, Johan Palmfeldt and Peter Bross declare that they have no conflict of interest.
Informed Consent
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5).
Details of the Contributions of Individual Authors
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1.
Paula Fernandez Guerra: experimental design, performance of experiments with fibroblasts from healthy individuals, data analysis and writing the first draft of the manuscript
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Martin Lund: performance of experiments with fibroblasts from patients
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Thomas Juhl Corydon: performance of confocal laser microscope experiments with MitoSOX
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Nanna Cornelius: performance of confocal laser microscope experiments with MitoSOX
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Niels Gregersen: VLCADD patient selection and hypothesis of oxidative stress in VLCADD patients
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Johan Palmfeldt: experimental design, report editing and data analysis
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7.
Peter Bross: experimental design, report editing and data analysis
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Fernandez-Guerra, P. et al. (2015). Application of an Image Cytometry Protocol for Cellular and Mitochondrial Phenotyping on Fibroblasts from Patients with Inherited Disorders. In: Morava, E., Baumgartner, M., Patterson, M., Rahman, S., Zschocke, J., Peters, V. (eds) JIMD Reports, Volume 27. JIMD Reports, vol 27. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8904_2015_494
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DOI: https://doi.org/10.1007/8904_2015_494
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