Advertisement

Large-Scale Mitotic Cell Synchronization

  • Kalyan Dulla
  • Anna SantamariaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 761)

Abstract

Understanding cell growth and cell division involves the study of regulatory events that occur in a cell cycle phase-dependent manner. Studies analyzing cell cycle regulatory mechanisms and cell cycle progression invariably require synchronization of cell populations at specific cell cycle stages. Several methods have been established to synchronize cells, including serum deprivation, contact inhibition, centrifugal elutriation, and drug-dependent synchronization. Despite potential adverse cellular consequences of synchronizing cells by pharmacological agents, drug-dependent methods can be advantageous when studying later cell cycle events to ensure specific enrichment at selected mitotic stages. This chapter describes protocols used in our laboratory for isolating mitotic mammalian cells in a large-scale manner. In particular, we discuss the technical aspects of adherent or suspension cell isolation, the methods necessary to enrich cells at different mitotic stages and the optimized culture conditions.

Key words

Mitosis large-scale synchronization HeLa S3 HeLa S spinner cultures triple flasks prometaphase metaphase anaphase/telophase 

Notes

Acknowledgments

We are thankful to Erich A. Nigg and Roman Körner for their support and critical reading of this chapter, and Herman H. Silljé, Albert Ries, and Elena Nigg for technical help to optimize these protocols. We apologize for any omission in references. We also acknowledge funding by the Biozentrum of the University of Basel, the Max Planck Society, and by ENFIN, funded by the European Commission within its FP6 Program.

References

  1. 1.
    Morgan, D. O. (2007) The cell cycle: principles of control. New Science Press, London.Google Scholar
  2. 2.
    Picotti, P., Bodenmiller, B., Mueller, L. N., Domon, B., and Aebersold, R. (2009) Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics, Cell 138, 795–806.PubMedCrossRefGoogle Scholar
  3. 3.
    Choudhary, C., and Mann, M. (2010) Decoding signalling networks by mass spectrometry-based proteomics, Nat. Rev. Mol. Cell Biol. 11, 427–439.PubMedCrossRefGoogle Scholar
  4. 4.
    Dephoure, N., Zhou, C., Villen, J., Beausoleil, S. A., Bakalarski, C. E., Elledge, S. J., et al. (2008) A quantitative atlas of mitotic phosphorylation, Proc. Natl. Acad. Sci. USA 105, 10762–10767.PubMedCrossRefGoogle Scholar
  5. 5.
    Nousiainen, M., Sillje, H. H., Sauer, G., Nigg, E. A., and Korner, R. (2006) Phosphoproteome analysis of the human mitotic spindle, Proc. Natl. Acad. Sci. USA 103, 5391–5396.PubMedCrossRefGoogle Scholar
  6. 6.
    Daub, H., Olsen, J. V., Bairlein, M., Gnad, F., Oppermann, F. S., Korner, R., et al. (2008) Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle, Mol. Cell 31, 438–448.PubMedCrossRefGoogle Scholar
  7. 7.
    Malik, R., Lenobel, R., Santamaria, A., Ries, A., Nigg, E. A., and Korner, R. (2009) Quantitative analysis of the human spindle phosphoproteome at distinct mitotic stages, J. Proteome Res. 8, 4553–4563.PubMedCrossRefGoogle Scholar
  8. 8.
    Dulla, K., Daub, H., Hornberger, R., Nigg, E. A., and Korner, R. (2010) Quantitative site-specific phosphorylation dynamics of human protein kinases during mitotic progression, Mol. Cell Proteomic. 9, 1167–1181.CrossRefGoogle Scholar
  9. 9.
    Holt, L. J., Tuch, B. B., Villen, J., Johnson, A. D., Gygi, S. P., and Morgan, D. O. (2009) Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution, Science 325, 1682–1686.PubMedCrossRefGoogle Scholar
  10. 10.
    Santamaria, A., Wang, B., Elowe, S., Malik, R., Zhang, F., Bauer, M., Schmidt, A., Silljé H. H., Körner, R, Nigg, E. A., (2011) The Plk1-dependent phosphoproteome of the early mitotic spindle, Mol. Cell Proteomic. 10(1): M110.004457.Google Scholar
  11. 11.
    Olsen, J. V., Vermeulen, M., Santamaria, A., Kumar, C., Miller, M. L., Jensen, L. J., et al. (2010) Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis, Sci. Signal. 3, ra3.PubMedCrossRefGoogle Scholar
  12. 12.
    Steen, J. A., Steen, H., Georgi, A., Parker, K., Springer, M., Kirchner, M., et al. (2008) Different phosphorylation states of the anaphase promoting complex in response to antimitotic drugs: a quantitative proteomic analysis, Proc. Natl. Acad. Sci. USA 105, 6069–6074.PubMedCrossRefGoogle Scholar
  13. 13.
    Terasima, T., and Tolmach, L. J. (1963) Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cells, Exp. Cell Res. 30, 344–362.PubMedCrossRefGoogle Scholar
  14. 14.
    Banfalvi, G. (2008) Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation, Nat. Protoc. 3, 663–673.PubMedCrossRefGoogle Scholar
  15. 15.
    Zieve, G. W., Turnbull, D., Mullins, J. M., and McIntosh, J. R. (1980) Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor nocodazole. Nocodazole accumulated mitotic cells, Exp. Cell Res. 126, 397–405.PubMedCrossRefGoogle Scholar
  16. 16.
    Ikegami, S., Taguchi, T., Ohashi, M., Oguro, M., Nagano, H., and Mano, Y. (1978) Aphidicolin prevents mitotic cell division by interfering with the activity of DNA polymerase-alpha, Nature 275, 458–460.PubMedCrossRefGoogle Scholar
  17. 17.
    Reichard, P., and Ehrenberg, A. (1983) Ribonucleotide reductase – a radical enzyme, Science 221, 514–519.PubMedCrossRefGoogle Scholar
  18. 18.
    Vassilev, L. T., Tovar, C., Chen, S., Knezevic, D., Zhao, X., Sun, H., et al. (2006) Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1, Proc. Natl. Acad. Sci. USA 103, 10660–10665.PubMedCrossRefGoogle Scholar
  19. 19.
    Ye, K., Ke, Y., Keshava, N., Shanks, J., Kapp, J. A., Tekmal, R. R., et al. (1998) Opium alkaloid noscapine is an antitumor agent that arrests metaphase and induces apoptosis in dividing cells, Proc. Natl. Acad. Sci. USA 95, 1601–1606.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Molecular DiagnosticsPhilips ResearchEindhovenThe Netherlands
  2. 2.BiozentrumUniversity of BaselBaselSwitzerland

Personalised recommendations