Cell sorting but not serum starvation is effective for SV40 human corneal epithelial cell cycle synchronization.
Cell sorting but not serum starvation is effective for SV40 human corneal epithelial cell cycle synchronization.
复制标题
细胞分选而非血清饥饿对于 SV40 人角膜上皮细胞周期同步有效。
DOI：
10.1016/j.exer.2005.11.007
复制
发表时间：
2006
影响因子：
3.4
通讯作者：
Murphy,ChristopherJ
中科院分区：
文献类型：
--
作者：
Liliensiek,SaraJ;Schell,Kathleen;Howard,Elise;Nealey,Paul;Murphy,ChristopherJ
SV 40人角膜上皮细胞（HCEC）群体容易用作难以在体外维持的原代角膜上皮细胞的替代物。为了用SV 40-HCEC细胞启动细胞周期实验，比较了两种单独的细胞同步化方法，包括血清饥饿和无菌细胞分选。我们假设，与其他同步方法相比，当进行细胞分选时，SV 40细胞以更高的效率同步进入每个细胞周期阶段（G1，S，G2 M）。通过在96小时内去除血清或用Höechst 33342染料标记使SV 40细胞同步化，并基于DNA含量进行分选。使用两种方法使细胞同步化，并在释放后最多72小时的时间点收获细胞。为了更精确地定义分选级分的性质，在分选之前用BrdU脉冲细胞。SV 40-HCEC细胞表现出明确的细胞周期谱。长达96小时的血清剥夺对于SV 40-HCEC的细胞同步化无效。相比之下，我们实现了SV 40-HCEC与无菌细胞分选的有效同步。在分选后，门控进入G1、S和G2 M的SV 40-HCEC细胞同步化高达85%，并保持同步化长达24小时。我们的研究结果表明，血清饥饿是无效的SV 40-HCEC细胞系的同步化。我们提出了一种更有效的方法，使用细胞分选的细胞同步化的SV 40-HCEC细胞。
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.