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 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 61-68 |
| Number of pages | 8 |
| Journal | Experimental Eye Research |
| Volume | 83 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jul 2006 |
| Externally published | Yes |
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Keywords
- cell cycle
- corneal epithelial cells
- flow cytometry
- synchronization
ASJC Scopus subject areas
- Ophthalmology
- Sensory Systems
Cite this
Cell sorting but not serum starvation is effective for SV40 human corneal epithelial cell cycle synchronization. / Liliensiek, Sara J.; Schell, Kathleen; Howard, Elise; Nealey, Paul; Murphy, Christopher J.
In: Experimental Eye Research, Vol. 83, No. 1, 07.2006, p. 61-68.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Cell sorting but not serum starvation is effective for SV40 human corneal epithelial cell cycle synchronization
AU - Liliensiek, Sara J.
AU - Schell, Kathleen
AU - Howard, Elise
AU - Nealey, Paul
AU - Murphy, Christopher J
PY - 2006/7
Y1 - 2006/7
N2 - 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.
AB - 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.
KW - cell cycle
KW - corneal epithelial cells
KW - flow cytometry
KW - synchronization
UR - http://www.scopus.com/inward/record.url?scp=33646894942&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33646894942&partnerID=8YFLogxK
U2 - 10.1016/j.exer.2005.11.007
DO - 10.1016/j.exer.2005.11.007
M3 - Article
VL - 83
SP - 61
EP - 68
JO - Experimental Eye Research
JF - Experimental Eye Research
SN - 0014-4835
IS - 1
ER -