Synchronization of Mammalian Cell Cultures by Serum Deprivation

Mammalian cells are amenable to the study of regulatory mechanisms dictating cell cycle progression in vitro by shifting them into the same phase of the cycle. Procedures to arrest cultured cells in specific phases of the cell cycle may be termed in vitro synchronization. The procedure described here was developed for the study of primary astrocytes and a glioma cell line, but is broadly applicable to other mammalian cells. Its application allows astrocytes to re-enter the cell cycle from a state of quiescence (G₀) under carefully defined experimental conditions to move together into subsequent phases such as the G₁ and S phases. A number of methods have been established to synchronize mammalian cell cultures, which include counterflow centrifugal elutriation, mitotic shake off, chemically induced cell cycle arrest, and newer live cell methods, such as cell permeable dyes. Yet, there are intrinsic limitations associated with these methods. In the present protocol, we describe a simple, reliable, and reversible procedure to synchronize astrocyte and glioma cultures from newborn rat brain by serum deprivation. The procedure is similar, and generally applicable, to other mammalian cells. This protocol consists essentially of two parts: (1) proliferation of astrocytes under optimal conditions in vitro until reaching desired confluence; and (2) synchronization and G₀ phase arrest of cultures by serum down-shift. This procedure has been utilized to examine cell cycle control in astroglioma cells and astrocytes from injured adult brain. It has also been employed in precursor cloning studies in developmental biology, suggesting wide applicability.