Application of an Image Cytometry Protocol for Cellular and Mitochondrial Phenotyping on Fibroblasts from Patients with Inherited Disorders

  • Paula Fernandez-GuerraEmail author
  • M. Lund
  • T. J. Corydon
  • N. Cornelius
  • N. Gregersen
  • J. Palmfeldt
  • Peter BrossEmail author
Research Report
Part of the JIMD Reports book series (JIMD, volume 27)


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.


Mitochondrial Membrane Potential Human Dermal Fibroblast H2O2 Treatment Acridine Orange Interassay Variation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



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.

Supplementary material

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(DOCX 596 kb)


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Copyright information

© SSIEM and Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Paula Fernandez-Guerra
    • 1
    Email author
  • M. Lund
    • 1
  • T. J. Corydon
    • 2
  • N. Cornelius
    • 1
    • 3
  • N. Gregersen
    • 1
  • J. Palmfeldt
    • 1
  • Peter Bross
    • 1
    Email author
  1. 1.Department of Clinical Medicine, Research Unit for Molecular Medicine (MMF)Aarhus University HospitalAarhusDenmark
  2. 2.Department of BiomedicineAarhus UniversityAarhusDenmark
  3. 3.Department of clinical Genetics, Applied Human Molecular GeneticsKennedy Center, Copenhagen University HospitalGlostrupDenmark

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