In vitro synchronization of mammalian astrocytic cultures by serum deprivation.
The study of the regulation of cell division cycle in vitro requires cell cultures growing in the same phase of the cycle. The procedure by which cells are arrested in specific phases of the cell cycle is termed synchronization. Synchronization is particularly important in the study of astrocyte biology, as its application allows astrocytes to re-enter the cell cycle from a state of quiescence (G(0)), and, under carefully defined experimental conditions, move together into subsequent phases such as the G(1) and S phases. A number of methods have been established to synchronize mammalian cell cultures, including centrifugal elutriation, mitotic shake-off, and 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 astrocytic cultures from newborn rat brains by serum deprivation. 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 down-shift and arrested in the G(0) phase of the cell cycle. This procedure has recently been extended toward the study of cell cycle control in astroglioma cells and astrocytes from injured adult brains. Since it was also employed in recent precursor cloning studies in developmental biology, this procedure will certainly find increasing use in future research.
Chou
Richard C
RC
Department of Neurology, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14214, USA.
Langan
Thomas J
TJ
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects*; D001973:Bromodeoxyuridine / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D004261:DNA Replication; D005455:Fluorescent Antibody Technique / Q000379:methods; D016193:G1 Phase; D005910:Glioma / Q000188:drug therapy / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D016196:S Phase; D012680:Sensitivity and Specificity; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors; D014316:Tritium / Q000378:metabolism; D014407:Tumor Cells, Cultured
D016895:Culture Media, Serum-Free; D014316:Tritium; D001973:Bromodeoxyuridine; D013936:Thymidine