Cell sorting but not serum starvation is effective for SV40 human corneal epithelial cell cycle synchronization

Abstract

SV40 human corneal epithelial cell (HCEC) populations are readily used as a substitute for primary corneal epithelial cells that are difficult to maintain in vitro. To initiate cell-cycle experiments with the SV40-HCEC cells, two separate methods of cell synchronization were compared including serum starvation and sterile cell sorting. We hypothesized that SV40 cells are synchronized at higher efficiencies into each cell cycle phase (G1, S, G2M) when cell sorting is performed when compared to alternative methods of synchronization. SV40 cells were synchronized by deprivation of serum over 96 h or labeled with Höechst 33342 dye and sorted based on DNA content. Cells were synchronized using both methods and harvested at time points up to 72 h after release. To define more precisely the nature of sorted fractions, cells were pulsed with BrdU prior to sorting. SV40-HCEC cells exhibit a well-defined cell cycle profile. Serum deprivation up to 96 h was ineffective for cell synchronization of SV40-HCECs. In comparison, we achieved efficient synchronization of the SV40-HCECs with sterile cell sorting. SV40-HCEC cells gated into G 1 , S and G 2 M were synchronized up to 85% following the sort and maintained synchronization up to 24 h. Our findings indicate that serum starvation is not effective for synchronization of the SV40-HCEC cell line. We present a more effective approach, the use of cell sorting for cell synchronization of the SV40-HCEC cells.

Semi-Mechanistic Pharmacokinetic/Pharmacodynamic Modeling of the Antitumor Activity of LY2835219, a New Cyclin-Dependent Kinase 4/6 Inhibitor, in Mice Bearing Human Tumor Xenografts

“…This is in agreement with previous reports of CDK4/6 inhibition leading to sequential emptying of the cell-cycle phases downstream of the restriction point, followed by an increasing spread of cells across phases upon release from synchronization (11,24,25). It should be pointed out, however, that using the same rate constant between each transit compartment would not have accounted for the different duration of each cell-cycle phase (17,26,27).…”

Characterization of Endothelial Basement Membrane Nanotopography in Rhesus Macaque as a Guide for Vessel Tissue Engineering

“…For example, feature sizes less than 200 nm promote increased adhesion and decreased proliferation in human corneal epithelial cells. 54,55 These studies illustrate that differences in the structure of the biophysical environment may be involved in guiding cell behavior and the importance of characterizing the basement membrane features for future scaffold design.…”

Aggressive breast cancer cells are dependent on activated Abl kinases for proliferation, anchorage-independent growth and survival

“…Sorted cells were rinsed with basal media, and plated on poly-D-lysine-coated dishes (60 mg/ml) in 0.1% FBS (Liliensiek et al, 2006). Attached cells were pretreated for 2 h with STI571, left unstimulated or stimulated with 5 nM IGF-1 or 10% FBS for the indicated times, labeled with BrdU, stained with fluorescein isothiocyanate-conjugated anti-BrdU antibody and propidium iodide (PI, 5 mg/ml) (BD Biosciences protocol, Chicago, IL, USA) and analysed by fluorescenceactivated cell sorting (FACS) using Cell Quest software (BD Biosciences) and Modfit analysis (Verity Software House, Topsham, ME, USA) (University of Kentucky Flow Cytometry Facility; MoFlo FACS; DAKO, Denmark).…”