Methods for Synchronizing Cells at Specific Stages of the Cell Cycle

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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 reversibly arrest cells at a particular cell cycle stage (e.g., lovastatin, see Basic Protocol 3; mimosine, see Alternate Protocol 4; and nocodazole, see Alternate Protocol 2). The protocols can be modified to enhance for mitotic cells (see Alternate Protocol 1) or to provide sequential G1/S blocks (see Alternate Protocol 5). Methods are provided for determining the mitotic index (see Support Protocol 1) and for measuring DNA synthesis by trichloroacetic acid (TCA) precipitation of [3H]thymidinelabeled DNA (see Support Protocol 2).

UNIT 8.3

thymidine; aphidicolin mimosine lovastatin G1 serum withdrawal; amino acid starvation mitotic shake-off nocodazole S

M

G2

Figure 8.3.1 Relationship between synchronization methods and cell cycle. Arrows indicate the point in the cell cycle at which cells are enriched by each method. Contributed by Joany Jackman and Patrick M. O’Connor Current Protocols in Cell Biology (1998) 8.3.1-8.3.20 Copyright © 1998 by John Wiley & Sons, Inc.

Cell Cycle Analysis

8.3.1

STRATEGIC PLANNING Prior to selecting a method of synchrony there are several factors to consider. Stage of Analysis Versus Stage of Synchrony In general, it is best to synchronize cells as close to the start of the cell cycle phase to be studied (Fig. 8.3.2). In most cases, cells will not maintain a high degree of synchrony through several rounds of cell division. In order to follow the expression of factors as a function of progression through a cell cycle phase, it is usually best to synchronize cells in the phase prior to the actual phase to be investigated. For example, to investigate the S phase–specific expression of a G1/S phase–cyclin protein (Gong et al., 1995), it may be necessary to synchronize cells in G1 or at the G1/S boundary using isoleucine deprivation or isoleucine deprivation coupled with aphidicolin treatment, respectively. The most difficult phase in which to investigate events related to both entry into and exit from is G1, mainly...
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