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

• Published in: Experimental eye research Vol. 83; no. 1; pp. 61 - 68
• Main Authors: Liliensiek, Sara J., Schell, Kathleen, Howard, Elise, Nealey, Paul, Murphy, Christopher J.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.07.2006
• Subjects: Benzimidazoles - analysis; Bromodeoxyuridine - analysis; cell cycle; Cell Cycle - physiology; Cell Line; Cell Separation; corneal epithelial cells; Culture Media, Serum-Free; DNA - analysis; Epithelial Cells - physiology; Epithelium, Corneal - cytology; flow cytometry; Flow Cytometry - methods; Fluorescent Dyes - analysis; G1 Phase - physiology; G2 Phase - physiology; Humans; Indicators and Reagents - analysis; Propidium - analysis; S Phase - physiology; Simian virus 40; synchronization; synchronization; flow cytometry; corneal epithelial cells; cell cycle
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2005.11.007

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 2M 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.