Large-Scale Mitotic Cell Synchronization
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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 wordsMitosis large-scale synchronization HeLa S3 HeLa S spinner cultures triple flasks prometaphase metaphase anaphase/telophase
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.
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