Synchronization of Mammalian Cells

Abstract

An essential and probably primary task in the study of cell cycle related processes is the synchronization of cells into specific cell cycle phases. Cell synchronization improves conditions by which an actual process under scrutiny can be studied and helps clarify linkage of the process to a particular cell cycle phase transition. In this chapter we will provide methods that have proven effective in synchronizing mammalian cells into defined cell cycle phases. We will discuss the advantages and disadvantages of each procedure and point to some key situations where such procedures have been employed successfully in the biochemical dissection of cell cycle control processes. Our focus here will be on the use of chemicals or growth restriction conditions to synchronize exponentially growing cells. We will present methodology to arrest cells in G0/G1, G1/S, G2 and mitosis-like states (Fig. 1). We will discuss the use of (1) serum starvation, isoleucine-withdrawal and chemicals (lovastatin, mimosine) for arrest of cells in a G0-like or G1-like state, (2) S phase blocking agents (aphidicolin, hydroxyurea, thymidine), (3) DNA damaging agents to produce G1 and/or G2 arrested cells, and (4) no- codazole to arrest cells in a mitotic state. Alternative nonchemical strategies using centrifugal elutriation as a basis for cell synchronization are discussed in the chapter by Marracino and Keng (Part One). Monitoring procedures to validate the success and selectivity of the arrest procedure, and subsequent cell cycle progression of the synchronize cohort will be discussed in the chapter by Giunta and Pucillo (Part One). Taken together, these chapters will appraise the currently available techniques that are commonly used for mammalian cell synchronization and provide the reader with methodology applicable to the study of a wide variety of cell cycle processes.