Advertisement

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)

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.

Keywords

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.

Notes

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.

Supplementary material

421110_1_En_494_MOESM1_ESM.docx (568 kb)
(DOCX 596 kb)

References

  1. Bie AS, Palmfeldt J, Hansen J et al (2011) A cell model to study different degrees of Hsp60 deficiency in HEK293 cells. Cell Stress Chaperones 16:633–640. doi: 10.1007/s12192-011-0275-5 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Brand MD, Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435:297–312. doi: 10.1113/expphysiol.2006.034330 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Burbulla LF, Krüger R (2012) The use of primary human fibroblasts for monitoring mitochondrial phenotypes in the field of Parkinson’s disease. J Vis Exp. doi: 10.3791/4228 PubMedPubMedCentralGoogle Scholar
  4. Burhans WC, Heintz NH (2008) The cell cycle is a redox cycle: linking phase-specific targets to cell fate. Free Radic Biol Med 47:1282–1293. doi: 10.1016/j.freeradbiomed.2009.05.026 CrossRefGoogle Scholar
  5. Cardoso AR, Kakimoto PA, Kowaltowski AJ (2013) Diet-sensitive sources of reactive oxygen species in liver mitochondria: role of very long chain acyl-CoA dehydrogenases. PLoS One 8:e77088. doi: 10.1371/journal.pone.0077088.g001 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Chan LL, Zhong X, Qiu J et al (2011) Cellometer vision as an alternative to flow cytometry for cell cycle analysis, mitochondrial potential, and immunophenotyping. Cytometry 79A:507–517. doi: 10.1002/cyto.a.21071 CrossRefGoogle Scholar
  7. Choi K, Kim J, Kim GW, Choi C (2009) Oxidative stress-induced necrotic cell death via mitochondria-dependent burst of reactive oxygen species. Curr Neurovasc Res 6:213–222CrossRefPubMedGoogle Scholar
  8. Cottet-Rousselle C, Ronot X, Leverve X, Mayol J-F (2011) Cytometric assessment of mitochondria using fluorescent probes. Cytometry A 79:405–425. doi: 10.1002/cyto.a.21061 CrossRefPubMedGoogle Scholar
  9. Dingley S, Chapman KA, Falk MJ (2011) Fluorescence-activated cell sorting analysis of mitochondrial content, membrane potential, and matrix oxidant burden in human lymphoblastoid cell lines. Methods Mol Biol 837:231–239. doi: 10.1007/978-1-61779-504-6_16 CrossRefGoogle Scholar
  10. Fernández-Guerra P, Birkler RID, Merinero B et al (2014) Selected reaction monitoring as an effective method for reliable quantification of disease-associated proteins in maple syrup urine disease. Mol Genet Genomic Med 2:383–392. doi: 10.1002/mgg3.88 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Freshney RI (2011) Introduction. Culture of animal cells. Wiley, Hoboken, pp 1–10Google Scholar
  12. Halter M (2012) Modernizing the MTT assay with microfluidic technology and image cytometry. Cytometry A 81:643–645. doi: 10.1002/cyto.a.22089 CrossRefPubMedGoogle Scholar
  13. Houten SM, Wanders RJA (2010) A general introduction to the biochemistry of mitochondrial fatty acid β-oxidation. J Inherit Metab Dis 33:469–477. doi: 10.1007/s10545-010-9061-2 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Jensen BC (2010) Skin deep: what can the study of dermal fibroblasts teach us about dilated cardiomyopathy? J Mol Cell Cardiol 48:576–578. doi: 10.1016/j.yjmcc.2009.11.021 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Lipman J, Flint O, Bradlaw J et al (1992) Cell culture systems and in vitro toxicity testing. Cytotechnology 8:129–176. doi: 10.1007/BF02525495 CrossRefGoogle Scholar
  16. Makpol S, Abdul Rahim N, Kien Hui C, Wan Ngah WZ (2012) Inhibition of mitochondrial cytochrome c release and suppression of caspases by gamma-tocotrienol prevent apoptosis and delay aging in stress-induced premature senescence of skin fibroblasts. Oxid Med Cell Longev 2012:1–13. doi: 10.1371/journal.pone.0004894 CrossRefGoogle Scholar
  17. Murphy MP (2009) How mitochondria produce reactive oxygen species. Biochem J 417:1. doi: 10.1042/BJ20081386 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Norberg E, Orrenius S, Zhivotovsky B (2010) Mitochondrial regulation of cell death: processing of apoptosis-inducing factor (AIF). Biochem Biophys Res Commun 396:95–100. doi: 10.1016/j.bbrc.2010.02.163 CrossRefPubMedGoogle Scholar
  19. Ozaki Y-I, Uda S, Saito TH et al (2010) A quantitative image cytometry technique for time series or population analyses of signaling networks. PLoS One 5:e9955. doi: 10.1371/journal.pone.0009955.t002 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Palmfeldt J, Vang S, Stenbroen V et al (2009) Mitochondrial proteomics on human fibroblasts for identification of metabolic imbalance and cellular stress. Proteome Sci 7:20. doi: 10.1186/1477-5956-7-20 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Perry S, Norman J, Barbieri J et al (2011) Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. Biotechniques 50:98–115. doi: 10.2144/000113610 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Phelan MC (1998) Basic techniques in mammalian cell tissue culture. Curr Protoc Cell Biol Chapter 1:Unit 1.1. doi: 10.1002/0471143030.cb0101s36
  23. Pierzchalski A, Mittag A, Tárnok A (2010) Introduction A: recent advances in cytometry instrumentation, probes, and methods--review. Methods Cell Biol 102:1–21. doi: 10.1016/B978-0-12-374912-3.00001-8 CrossRefGoogle Scholar
  24. Rittié L, Fisher GJ (2005) Isolation and culture of skin fibroblasts. Methods Mol Med 117:83–98. doi: 10.1385/1-59259-940-0:083 PubMedGoogle Scholar
  25. Robinson KM, Janes MS, Beckman JS (2007) The selective detection of mitochondrial superoxide by live cell imaging. Nat Protoc 3:941–947. doi: 10.1038/nprot.2008.56 CrossRefGoogle Scholar
  26. Sandell L, Sakai D (2011) Mammalian cell culture. Curr Protoc Essent Lab Tech 4.3. 1–4.3. 32Google Scholar
  27. Schiff M, Mohsen A-W, Karunanidhi A et al (2013) Molecular and cellular pathology of very-long-chain acyl-CoA dehydrogenase deficiency. Mol Genet Metab 109:21–27. doi: 10.1016/j.ymgme.2013.02.002 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Skindersoe ME, Rohde M, Kjaerulff S (2012) A novel and rapid apoptosis assay based on thiol redox status. Cytometry 81A:430–436. doi: 10.1002/cyto.a.22032 CrossRefGoogle Scholar
  29. Smith CL (2006) Mammalian cell culture. Curr Protoc Cell Biol Chapter 28:Unit 0.1. doi: 10.1002/0471142727.mb2800s73
  30. Tucci S, Primassin S, Spiekerkoetter U (2010) Fasting-induced oxidative stress in very long chain acyl-CoA dehydrogenase-deficient mice. FEBS J 277:4699–4708. doi: 10.1111/j.1742-4658.2010.07876.x CrossRefPubMedGoogle Scholar
  31. Valko M, Leibfritz D, Moncol J et al (2006) Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39:44–84. doi: 10.1016/j.biocel.2006.07.001 CrossRefPubMedGoogle Scholar
  32. Wang Y, Mohsen A-W, Mihalik SJ et al (2010) Evidence for physical association of mitochondrial fatty acid oxidation and oxidative phosphorylation complexes. J Biol Chem 285:29834–29841. doi: 10.1074/jbc.M110.139493 CrossRefPubMedPubMedCentralGoogle Scholar

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

Personalised recommendations