Synchronization of Mammalian Cell Cultures by Serum Deprivation

  • Thomas J. LanganEmail author
  • Richard C. Chou
Part of the Methods in Molecular Biology book series (MIMB, volume 761)


Mammalian cells are amenable to study the regulation of cell cycle progression in vitro by shifting them into the same phase of the cycle. Procedures to arrest cultured cells in specific phases of the cell cycle may be termed in vitro synchronization. The procedure described here was developed for the study of primary astrocytes and a glioma cell line, but is applicable to other mammalian cells. Its application allows astrocytes to reenter the cell cycle from a state of quiescence (G0), and then, under carefully defined experimental conditions, to move together into subsequent phases such as the G1 and S phases. A number of methods have been established to synchronize mammalian cell cultures, which include physical separation by centrifugal elutriation and mitotic shake off or chemically induced cell cycle arrest. Yet, there are intrinsic limitations associated with these methods. In the present protocol, we describe a simple, reliable, and reversible procedure to synchronize astrocyte and glioma cultures from newborn rat brain by serum deprivation. The procedure is similar, and generally applicable, to other mammalian cells. This protocol consists essentially of two parts: (1) proliferation of astrocytes under optimal conditions in vitro until reaching desired confluence; and (2) synchronization of cultures by serum downshift and arrested in the G0 phase of the cell cycle. This procedure has been extended to the examination of cell cycle control in astroglioma cells and astrocytes from injured adult brain. It has also been employed in precursor cloning studies in developmental biology, suggesting wide applicability.

Key words

G0 Astrocytes Alzheimer’s disease cell cycle glioma oncogenesis synchronization serum deprivation 


  1. 1.
    Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. (2002) Molecular biology of the cell. Fourth edition,  Chap. 17. The cell cycle and programmed cell death, pp. 983–1026. Garland Science, NY.Google Scholar
  2. 2.
    Ashihara, T., and Baserga, R. (1979) Cell synchronization. Methods Enzymol. 58, 248–262.PubMedCrossRefGoogle Scholar
  3. 3.
    Bartholomew, J. C., Neff, N. T., and Ross, P. A. (1976) Stimulation of WI-38 cell cycle transit: effect of serum concentration and cell density. J. Cell. Physiol. 89, 251–258.PubMedCrossRefGoogle Scholar
  4. 4.
    Campbell, A. (1957) Synchronization of cell division. Bacteriol. Rev. 21, 263–272.PubMedGoogle Scholar
  5. 5.
    Salomoni, P., and Callegari, F. (2010) Cell cycle control of mammalian neural stem cells: putting a speed limit on G1. Trends Cell Biol. 20, 233–243.PubMedCrossRefGoogle Scholar
  6. 6.
    Wang, W., Bu, B., Zhang, M., Yu, Z., and Tao, D. (2009) Neural cell cycle dysregulation and central nervous system diseases. Progr Neurobiol. 89, 1–17.CrossRefGoogle Scholar
  7. 7.
    Pardee, A. B. (1974) A restriction point for control of normal animal cell proliferation. Proc. Natl. Acad. Sci. USA 71, 1286–1290.PubMedCrossRefGoogle Scholar
  8. 8.
    Langan, T. J., and Volpe, J. J. (1986) Obligatory relationship between the sterol biosynthetic pathway and DNA synthesis and cell proliferation in glial primary cultures. J. Neurochem. 46, 1283–1291.PubMedCrossRefGoogle Scholar
  9. 9.
    Li, V., Kelly, K., Schrot, R., and Langan, T. J. (1996) Cell cycle kinetics and commitment in newborn, adult, and tumoral astrocytes. Brain Res. 96, 138–147.CrossRefGoogle Scholar
  10. 10.
    Quesney-Huneeus, V., Galick, H. A., Siperstein, M. D., Erickson, S. K., Spencer, T. A., and Nelson, J. A. (1983) The dual role of mevalonate in the cell cycle. J. Biol. Chem. 258, 378–385.PubMedGoogle Scholar
  11. 11.
    Merrill, G. F. (1998) Cell synchronization. In J. P. Mather and D. Barnes (Eds.), Methods in cell biology, Vol. 57, pp. 229–249. Academic Press, San Diego.Google Scholar
  12. 12.
    Keyomarsi, K., Sandoval, L., Band, V., and Pardee, A. B. (1991) Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin. Cancer Res. 51, 3602–3609.PubMedGoogle Scholar
  13. 13.
    Krek, W., and DeCaprio, J. A. (1995) Cell synchronization. Methods Enzymol. 254, 114–124.PubMedCrossRefGoogle Scholar
  14. 14.
    Pardee, A. B., and Keyomarsi, K. (1992) Modification of cell proliferation with inhibitors. Curr. Opin. Cell Biol. 4, 186–191.PubMedCrossRefGoogle Scholar
  15. 15.
    Langan, T. J., and Slater, M. C. (1991) Quiescent astroglia in long-term primary cultures re-enter the cell cycle and require a nonsterol isoprenoid in late G1. Brain Res. 548, 9–17.PubMedCrossRefGoogle Scholar
  16. 16.
    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
  17. 17.
    Johnston, D. A., White, R. A., and Barlogie, B. A. (1978) Automatic processing and interpretation of DNA distributions: comparison of several techniques. Comp. Biomed. Res. 11, 393–404.CrossRefGoogle Scholar
  18. 18.
    Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J., and Campbell, K. H. S. (1997) Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–813.PubMedCrossRefGoogle Scholar
  19. 19.
    Sanchez, I., Goya, L., Vallerga, A. K., and Firestone, G. L. (1993) Glucocorticoids reversibly arrest rat hepatoma cell growth by inducing an early G1 block in cell cycle progression. Cell Growth Differ. 4, 215–225.PubMedGoogle Scholar
  20. 20.
    Huberman, J. A. (1981) New views of the biochemistry of eucaryotic DNA replication revealed by aphidicolin, an unusual inhibitor of DNA polymerase alpha. Cell 23, 647–648.PubMedCrossRefGoogle Scholar
  21. 21.
    Mitchell, B. F., and Tupper, J. T. (1977) Synchronization of mouse 3T3 and SV40 3T3 cells by way of centrifugal elutriation. Exp. Cell Res. 106, 351–355.PubMedCrossRefGoogle Scholar
  22. 22.
    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
  23. 23.
    Webber, L. M., and Garson, O. M. (1983) Fluorodeoxyuridine synchronization of bone marrow cultures. Cancer Genet. Cytogenet. 8, 123–132.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Neurology, Pediatrics, Physiology and BiophysicsSchool of Medicine and Biomedical Sciences, Children’s Hospital, State University of New YorkBuffaloUSA
  2. 2.Dartmouth School of MedicineLebanonUSA

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