Abstract
The method described in the following chapter utilizes a double thymidine block (an inhibitor of DNA synthesis) followed by treatment of cells with nocodazole (a mitotic inhibitor) to obtain large cell populations at distinct phases of the cell cycle. Treatment with double thymidine results in a G1/S-phase arrested cell population, and the use of flow cytometry allows progression of the cells through the cell cycle to be monitored. Flow cytometry enables the calculation of timings for collection of cells at distinct cell cycle phases from G1/S (following treatment with thymidine) through to G2/M (owing to the presence of nocodazole).
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References
Hyland, P. L., Keegan, A. L., Curran, M. D., Middleton, D., McKenna, P. G., and Barnett, Y. A. (2000) Effect of a dCTP:dTTP pool imbalance on DNA replication fidelity in Friend murine erythroleukemia cells. Environ. Mol. Mutagen. 36, 87–96.
Jordan, M. A., Thrower, D., and Wilson, L. (1992) Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis. J. Cell Sci. 102, 401–416.
Vasquez, R. J., Howell, B., Yvon, A. M., Wadsworth, P., and Cassimeris, L. (1997) Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro. Mol. Biol. Cell. 8, 973–985.
O’Connor, P. M. and Jackman, J. (1995) Synchronisation of mammalian cells, in Cell Cycle-Materials and Methods (Pagano, M. ed.), Springer-Verlag, New York, pp. 61–74.
Lee, K. S., Yuan, Y-L. O., Kuriyama, R., and Erikson, R. L. (1995) Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1. Mol. Cell. Biol. 15, 7143–7151.
Tas, J., and Westerneng, G. J. (1981) Fundamental aspects of the interaction of propidium diiodide with nuclei acids studied in a model system of polyacrylamide films. Histochem. Cytochem. 29, 929–936.
Pagano, M., Pepperkok, R., Verde, F., Ansorge, W. and Draetta, G. (1992) Cyclin A is required at two points in the human cell cycle. EMBO J. 11, 961–971.
Murray, A. W. and Kirschner, M. W. (1989) Cyclin synthesis drives the early embryonic cell cycle. Nature 339, 275–280.
Dulic, V., Lees, E., and Reed, S. I. (1992) Association of human cyclin E with a periodic G1-S phase protein kinase. Science 257, 1958–1961.
Koff, A., Giordano, A., Desai, D., et al. (1992) Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. Science 257, 1689–1694.
Ohtsubo, M. and Roberts, J. M. (1993) Cyclin-dependent regulation of G1 in mammalian fibroblasts. Science 259, 1908–1912.
Tsai, L. H., Lees, E., Faha, B., Harlow, E., and Riabowol, K. (1993) The cdk2 kinase is required for the G1-to-S transition in mammalian cells. Oncogene 8, 1593–1602.
Resnitzky, D., Gossen, M., Bujard, H., and Reed, S. I. (1994) Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol. Cell. Biol. 14, 1669–1679.
Lacey, K. R., Jackson, P. K., and Stearns, T. (1999) Cyclin-dependent kinase control of centrosome duplication. Proc. Natl. Acad. Sci. USA 96, 2817–2822.
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Harper, J.V. (2005). Synchronization of Cell Populations in G1/S and G2/M Phases of the Cell Cycle. In: Humphrey, T., Brooks, G. (eds) Cell Cycle Control. Methods in Molecular Biology™, vol 296. Humana Press. https://doi.org/10.1385/1-59259-857-9:157
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DOI: https://doi.org/10.1385/1-59259-857-9:157
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