Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
The protocols presented here describe procedures used to synchronize cells in various stages of the cell cycle (Fig. 8.3.1). Synchronization is particularly useful for investigating a particular cell cycle–regulated event or preparing cells for extraction of transient factors whose expression is dependent on cell cycle stage. Exponentially growing cultures are generally asynchronous; i.e., each cell progresses through the cell cycle independently of the cell cycle stage of its neighboring cells. Cells that are synchronized are artificially induced to cycle in a homogeneous manner. The ability to continue cycling is an important distinction between a homogeneous population of cells created by synchronization and one created by blocking cells from cycling. Blocking cells from cycling may also result in a homogeneous population of cells at a particular stage of the cell cycle, but often results in death of the cell. In contrast, the purpose of synchonization is to create an enriched population of cells at a single stage of the cell cycle; these cells will then be able to continue through the cell cycle with as little disruption of normal events as possible. For a comprehensive review of events and explanation of the salient features of each of these stages see Pines (1995), Hartwell and Kastan (1994), UNIT 8.1, and the chapter introduction. Techniques will be presented for synchronizing cells in the G1, S, and M phases of the cell cycle. These techniques include a selection of methodologies that capitalize on the biology and biochemistry of eukaryotic cells, such as selective nutrient depletion (e.g., isoleucine deprivation, see Alternate Protocol 3; and serum withdrawal, see Basic Protocol 2), feedback control through addition of excess nutrients (e.g., thymidine, see Basic Protocol 4), morphological differences (e.g., mitotic shake-off, see Basic Protocol 1), or the use of chemical agents to
The protocols presented here describe procedures used to synchronize cells in various stages of the cell cycle (Fig. 8.3.1). Synchronization is particularly useful for investigating a particular cell cycle–regulated event or preparing cells for extraction of transient factors whose expression is dependent on cell cycle stage. Exponentially growing cultures are generally asynchronous; i.e., each cell progresses through the cell cycle independently of the cell cycle stage of its neighboring cells. Cells that are synchronized are artificially induced to cycle in a homogeneous manner. The ability to continue cycling is an important distinction between a homogeneous population of cells created by synchronization and one created by blocking cells from cycling. Blocking cells from cycling may also result in a homogeneous population of cells at a particular stage of the cell cycle, but often results in death of the cell. In contrast, the purpose of synchonization is to create an enriched population of cells at a single stage of the cell cycle; these cells will then be able to continue through the cell cycle with as little disruption of normal events as possible. For a comprehensive review of events and explanation of the salient features of each of these stages see Pines (1995), Hartwell and Kastan (1994), UNIT 8.1, and the chapter introduction. Techniques will be presented for synchronizing cells in the G1, S, and M phases of the cell cycle. These techniques include a selection of methodologies that capitalize on the biology and biochemistry of eukaryotic cells, such as selective nutrient depletion (e.g., isoleucine deprivation, see Alternate Protocol 3; and serum withdrawal, see Basic Protocol 2), feedback control through addition of excess nutrients (e.g., thymidine, see Basic Protocol 4), morphological differences (e.g., mitotic shake-off, see Basic Protocol 1), or the use of chemical agents to