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Free keywords:
QUANTITATIVE PHOSPHOPROTEOMICS; PHOSPHORYLATION; CYCLE; CDK1Biochemistry & Molecular Biology; Cell Biology; Mitosis; Large-scale synchronization; HeLa S3; HeLa S; Spinner cultures;
Triple flasks; Prometaphase; Metaphase; Anaphase/telophase;
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.
