Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.prot4489

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Synchronization of Mammalian Cell Cultures in Mitosis Using Selective Detachment

Paul J. Galgano and Carl L. Schildkraut

This protocol was adapted from "Cell Synchronization," Chapter 14, in Cells (eds. Spector et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. This three-volume set is now out of print; however, some of the microscopy methods were republished in Basic Methods in Microscopy, by David L. Spector and Robert D. Goldman.


INTRODUCTION

This protocol describes a method for synchronizing monolayer cells in mitosis using selective detachment from their substrate. During mitosis, cells become more spherical, causing them to become more loosely attached to their substrate. The "rounded up" cells are selectively detached by tapping the culture flask, resulting in a population in which as many as 90-98% of the cells are in mitosis. The drug nocodazole is used to increase the percentage of cells undergoing mitosis before detachment is performed. This procedure has been applied to mouse fibroblast and CHO (Chinese hamster ovary) cells. Since different cell types may attach differently, it will be necessary to determine the amount of force needed to remove loosely attached cells and the mitotic cells.


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