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Methods for Cell Sorting of Young and Senescent Cells

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Biological Aging

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 371))

Abstract

Cellular senescence, the ultimate and irreversible loss of replicative capacity of cells in primary culture, has been a popular model for studying the aging process. However, the replicative life span of human fibroblasts is heterogeneous even in clonal populations, with the fraction of senescent cells increasing at each population doubling, rather than all cells entering senescence simultaneously. Thus, the study of individual cells in a mass culture is of extreme importance to the understanding of replicative senescence. Cell sorting is a method that allows physical separation of cells with different characteristics when measured by flow cytometry. Here, we describe various methods by which cells that reach senescence early can be physically sorted out of a bulk of growing cells, and discuss how different methods can affect the posterior analysis of the sorted populations.

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References

  1. Smith, J. R. and Whitney, R. G. (1980) Intraclonal variation in proliferative potential of human diploid fibroblasts: stochastic mechanism for cellular aging. Science 207, 82–84.

    Article  CAS  PubMed  Google Scholar 

  2. Kill, I. R., et al. (1994) The expression of proliferation-dependent antigens during the lifespan of normal and progeroid human fibroblasts in culture. J. Cell Sci. 107 (Pt 2), 571–579.

    CAS  PubMed  Google Scholar 

  3. Thomas, E., et al. (1997) Different kinetics of senescence in human fibroblasts and peritoneal mesothelial cells. Exper. Cell Res. 236, 355–358.

    Article  CAS  Google Scholar 

  4. Bond, J. A., Wyllie, F. S., and Wynford-Thomas, D. (1994) Escape from senescence in human diploid fibroblasts induced directly by mutant p53. Oncogene 9, 1885–1889.

    CAS  PubMed  Google Scholar 

  5. Fagagna, F.d.A.d., et al. (2003) A DNA damage checkpoint response in telomereinitiated senescence. Nature 426, 194–198.

    Article  Google Scholar 

  6. Martin-Ruiz, C., et al. (2004) Stochastic variation in telomere shortening rate causes heterogeneity of human fibroblast replicative life span. J. Biol. Chem. 279, 17,826–17,833.

    Article  CAS  PubMed  Google Scholar 

  7. Herbig, U., et al. (2004) Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a Mol. Cell. 14, 501–513.

    Article  CAS  PubMed  Google Scholar 

  8. Zglinicki, T. V., et al. (2005) Human cell senescence as a DNA damage response. Mech. Ageing Dev. 126, 111–117.

    Article  Google Scholar 

  9. Sgonc, R. and Gruber, J. (1998) Apoptosis detection: an overview. Exp. Gerontol. 33, 525–533.

    Article  CAS  PubMed  Google Scholar 

  10. Wei, W. and Sedivy, J. M. (1999) Differentiation between Senescence (M1) and Crisis (M2) in Human Fibroblast Cultures. Exp. Cell Res. 253, 519–522.

    Article  CAS  PubMed  Google Scholar 

  11. Bayreuther, K., et al. (1988) Differentiation of fibroblast stem cells. J. Cell Sci. Suppl. 10, 115–130.

    CAS  PubMed  Google Scholar 

  12. Gorbunova, V., Seluanov, A., and Pereira-Smith, O. M. (2003) Evidence that high telomerase activity may induce a senescent-like growth arrest in human fibroblasts. J. Biol. Chem. 278, 7692–7698.

    Article  CAS  PubMed  Google Scholar 

  13. Sitte, N., et al. (2001) Lipofuscin accumulation in proliferating fibroblasts in vitro: an indicator of oxidative stress. Exp. Gerontol. 36, 475–486.

    Article  CAS  PubMed  Google Scholar 

  14. Hutter, E., et al. (2002) Replicative senescence of human fibroblasts: the role of Ras-dependent signaling and oxidative stress. Exp. Gerontol. 37, 1165–1174.

    Article  CAS  PubMed  Google Scholar 

  15. Allen, R. G., et al. (1999) Differences in electron transport potential, antioxidant defenses, and oxidant generation in young and senescent fetal lung fibroblasts (WI-38). J. Cell Physiol. 180, 114–122.

    Article  CAS  PubMed  Google Scholar 

  16. Santos, J. H., et al. (2003) Cell sorting experiments link persistent mitochondrial DNA damage with loss of mitochondrial membrane potential and apoptotic cell death. J. Biol. Chem. 278, 1728–1734.

    Article  CAS  PubMed  Google Scholar 

  17. Scholzen, T. and Gerdes, J. (2000) The Ki-67 protein: from the Known and the Unknown. J. Cell. Physiol. 182, 311–322.

    Article  CAS  PubMed  Google Scholar 

  18. Satyanarayana, A., et al. (2004) Mitogen stimulation cooperates with telomere shortening to activate DNA damage responses and senescence signaling. Mol. Cell. Biol. 24, 5459–5474.

    Article  CAS  PubMed  Google Scholar 

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© 2007 Humana Press Inc., Totowa, NJ

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Passos, J.F., von Zglinicki, T. (2007). Methods for Cell Sorting of Young and Senescent Cells. In: Tollefsbol, T.O. (eds) Biological Aging. Methods in Molecular Biology™, vol 371. Humana Press. https://doi.org/10.1007/978-1-59745-361-5_4

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  • DOI: https://doi.org/10.1007/978-1-59745-361-5_4

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-658-0

  • Online ISBN: 978-1-59745-361-5

  • eBook Packages: Springer Protocols

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