Document Detail

Cell sorting but not serum starvation is effective for SV40 human corneal epithelial cell cycle synchronization.
MedLine Citation:
PMID:  16545370     Owner:  NLM     Status:  MEDLINE    
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 G1, S and G2M 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.
Sara J Liliensiek; Kathleen Schell; Elise Howard; Paul Nealey; Christopher J Murphy
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2006-03-20
Journal Detail:
Title:  Experimental eye research     Volume:  83     ISSN:  0014-4835     ISO Abbreviation:  Exp. Eye Res.     Publication Date:  2006 Jul 
Date Detail:
Created Date:  2006-06-05     Completed Date:  2006-08-07     Revised Date:  2014-09-15    
Medline Journal Info:
Nlm Unique ID:  0370707     Medline TA:  Exp Eye Res     Country:  England    
Other Details:
Languages:  eng     Pagination:  61-8     Citation Subset:  IM    
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MeSH Terms
Benzimidazoles / analysis
Bromodeoxyuridine / analysis
Cell Cycle / physiology*
Cell Line
Cell Separation
Culture Media, Serum-Free
DNA / analysis
Epithelial Cells / physiology
Epithelium, Corneal / cytology
Flow Cytometry / methods*
Fluorescent Dyes / analysis
G1 Phase / physiology
G2 Phase / physiology
Indicators and Reagents / analysis
Propidium / analysis
S Phase / physiology
Simian virus 40
Grant Support
CA14520/CA/NCI NIH HHS; R01 EY012253/EY/NEI NIH HHS; R01 EY012253-02/EY/NEI NIH HHS; R01 EY016134/EY/NEI NIH HHS; R01 EY016134-01A2/EY/NEI NIH HHS; R01 NEI12253-7//PHS HHS
Reg. No./Substance:
0/Benzimidazoles; 0/Culture Media, Serum-Free; 0/Fluorescent Dyes; 0/Indicators and Reagents; 23491-52-3/HOE 33342; 36015-30-2/Propidium; 9007-49-2/DNA; G34N38R2N1/Bromodeoxyuridine
Erratum In:
Exp Eye Res. 2006 Dec;83(6):1521

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