|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Modification of the alkaline comet assay with human mesenchymal stem cells
Robert Fuchs*12, Ingeborg Stelzer*†1, Christoph M. P. Drees*†, Christian Rehnolt*†, Elisabeth Schraml‡, Anton Sadjak* and Wolfgang Schwinger§
*Institute of Pathophysiology and Immunology, Center of Molecular Medicine, Medical University of Graz, Heinrichstrasse 31A, 8010 Graz, Austria, †Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria, ‡Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria, and §Division of Pediatric HematologyOncology, Department of Pediatrics and Adolescent Medicine, Medical University of Graz, Auenbruggerplatz 30, 8036 Graz, Austria
MSCs (mesenchymal stem cells) are planned foruse in regenerative medicine to offset age-dependent alterations. However, MSCs are affected by replicative senescence associated with decreasing proliferation potential, telomere shortening and DNA damage during in vitro propagation. To monitor in vitro senescence, we have assessed the integrity of DNA by the alkaline comet assay. For optimization of the comet assay we have enhanced the stability of comet slides in liquid and minimized the background noise of the method by improving adhesion of agarose gels on the comet slides and concentrating cells on a defined small area on the slides. The modifications of the slide preparation increase the overall efficiency and reproducibility of the comet assay and minimize the image capture and storage. DNA damage of human MSCs during in vitro cultivation increased with time, as assessed by the comet assay, which therefore offers a fast and easy screening tool in future efforts to minimize replicative senescence of MSCs in vitro.
Key words: comet assay, DNA damage; human mesenchymal stem cell (MSC), replicative senescence
Abbreviations: BM, bone marrow, DAPI, 4′,6-diamidino-2-phenylindol, FBS, foetal bovine serum, MSC, mesenchymal stem cells, MNC, mononuclear cells, ROS, reactive oxygen species
1These authors contributed equally to this study.
2To whom correspondence should be addressed (email firstname.lastname@example.org).
Received 2 May 2011/20 July 2011; accepted 16 September 2011
Published as Cell Biology International Immediate Publication 16 September 2011, doi:10.1042/CBI20110251
© The Author(s) Journal compilation © 2012 Portland Press Limited