Synchronization of Mammalian Cell Cultures by Serum Deprivation

Abstract

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 (G) under carefully defined experimental conditions to move together into subsequent phases such as the G 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 G 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.

Two-way communication between the metabolic and cell cycle machineries: the molecular basis

“…Accordingly, in the absence of mitogens, even in a nutrient-rich environment, cells will not enter the cell cycle 23 . On the other hand, even in the presence of promitogenic cues, glucose deprivation will keep cells from proliferating, which is a widely used method for synchronizing mammalian cells 24 . The fact that signaling pathways coordinating cell cycle progression control, and are controlled by, changes in cellular metabolism 25,26 shows that, also in multicellular organisms, there must be a crosstalk between these pathways, cell cycle and metabolism.…”

Peer Review #2 of "Albumin promotes proliferation of G1 arrested serum starved hepatocellular carcinoma cells (v0.1)"

“…They argued that albumin inhibits tumour evidenced by its increased levels correlating with cancer remission and addition of albumin to serum starved hepatoma cells caused G1 arrest (Nojiri & Joh, 2014;Bağırsakçı et al, 2017). It is worth noting that serum is essential for hepatocellular carcinoma growth (Zhuge & Cederbaum, 2006;Liang et al, 2013) whereas serum starvation is routinely carried out to arrest cells in G1 phase (Davis, Ho & Dowdy, 2001;Langan, Rodgers & Chou, 2017). These observations that albumin causes cell cycle arrest are possibly a result of an artefact.…”

Growth Factors Delivery System for Skin Regeneration: An Advanced Wound Dressing

“…To synchronize the cells before to start the treatments, BMSC were placed in SFM for 24, 48 and 72 h [22]. To evaluate the residual proliferation of BMSC, a Bromodeoxyuridine (BrdU) assay (Roche, Basel, Switzerland) was performed according to the manufacturer's instructions.…”