Protocol

G₁ Synchronization of CHO Cells by Isoleucine Deprivation

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 provides a method for the synchronization of a monolayer culture of CHO cells in G₁ using isoleucine deprivation. Since CHO cells can also be adapted to grow in suspension culture, this procedure can be used to obtain larger quantities of cells. When isoleucine is replaced, the cells resume growth and begin to enter S phase ~4 hours later. This method arrests almost 100% of the CHO cells in G₁, and upon reversal, leads to rapid recovery of cell growth and very high cell viability. This method has been used with other hamster cell lines, some transformed mouse cells, and some transformed human cell lines have also been used, but with varying effectiveness.