Protocol

Cell Cycle Synchronization

Volume 761 of the series Methods in Molecular Biology pp 75-83

Date:

Synchronization of Mammalian Cell Cultures by Serum Deprivation

  • Thomas J. LanganAffiliated withDepartment of Neurology, Pediatrics, Physiology and Biophysics, School of Medicine and Biomedical Sciences, Children’s Hospital, State University of New York Email author 
  • , Richard C. ChouAffiliated withDartmouth School of Medicine

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Abstract

Mammalian cells are amenable to study the regulation of 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 applicable to other mammalian cells. Its application allows astrocytes to reenter the cell cycle from a state of quiescence (G0), and then, 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 physical separation by centrifugal elutriation and mitotic shake off or chemically induced cell cycle arrest. 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 of cultures by serum downshift and arrested in the G0 phase of the cell cycle. This procedure has been extended to the examination of 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.

Key words

G0 Astrocytes Alzheimer’s disease cell cycle glioma oncogenesis synchronization serum deprivation