A Critical Appraisal of Synchronization Methods Applied to Achieve Maximal Enrichment of HeLa Cells in Specific Cell Cycle Phases

Knehr, Michael; Poppe, Michael; Enulescu, M.; Eickelbaum, W.; Stoehr, Michael; Schroeter, D.; Paweletz, Neidhard

doi:10.1006/excr.1995.1121

Cited by 47 publications

(38 citation statements)

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“…3B) and the cell cycle of HeLa cells is well characterized. [15][16][17] Our results strongly suggest that CAC1 is a cell cycle associated protein capable of promoting cell proliferation through the activation of CDK2 at the G 1 /S phase transition.…”

Section: Introductionmentioning

confidence: 57%

Identification and characterization of CAC1 as a novel CDK2-associated cullin

Kong

Kang²,

Yin³

2009

Cell Cycle

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Section: Introductionmentioning

confidence: 57%

Identification and characterization of CAC1 as a novel CDK2-associated cullin

Kong

Kang²,

Yin³

2009

Cell Cycle

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“…Synchronization at the G1/S border was performed on non con¯uent HeLa cell cultures by the double thymidine block (DTB) method (Knehr et al, 1995). After 8 h of incubation, cells were in G2.…”

Section: Cell Synchronizationmentioning

confidence: 99%

The bacterial cytolethal distending toxin (CDT) triggers a G2 cell cycle checkpoint in mammalian cells without preliminary induction of DNA strand breaks

et al. 1999

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The bacterial cytolethal distending toxin (CDT) was previously shown to arrest the tumor-derived HeLa cell line in the G2-phase of the cell cycle through inactivation of CDK1, a cyclin-dependent kinase whose state of activation determines entry into mitosis. We have analysed the e ects induced in HeLa cells by CDT, in comparison to those induced by etoposide, a prototype anti-tumoral agent that triggers a G2 cell cycle checkpoint by inducing DNA damage. Both CDT and etoposide inhibit cell proliferation and induces the formation of enlarged mononucleated cells blocked in G2. In both cases, CDK1 from arrested cells could be reactivated both in vitro by dephosphorylation by recombinant Cdc25B phosphatase and in vivo by ca eine. However, the cell cycle arrest triggered by CDT, unlike etoposide, did not originate from DNA strand breaks as demonstrated in the single cell gel electrophoresis assay and by the absence of slowing down of S phase in synchronized cells. Together with additional observations on synchronized HeLa cells, our results suggest that CDT triggers a G2 cell cycle checkpoint that is initiated during DNA replication and that is independent of DNA damage.

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“…Serum starvation can have many negative consequences including induction of DNA fragmentation (Niemann et al, 2000), decreased mitochondrial viability (Takeda et al, 2002), alterations or disruption of the cell cycle following serum starvation (Gray and Darzynkiewicz, 1987) and the induction of apoptosis or death (Magnani and Bettini, 2000;Oya et al, 2003;Tan et al, 2003). In addition, some transformed and primary cell lines, including primary rabbit corneal epithelial cells are resistant to the effects of serum starvation and will not synchronize with this method (Knehr et al, 1995;Cooper, 1998;Anderson and SundarRaj, 2001;Katska et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

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

Liliensiek¹,

Schell²,

Howard³

et al. 2006

Experimental Eye Research

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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.

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A Critical Appraisal of Synchronization Methods Applied to Achieve Maximal Enrichment of HeLa Cells in Specific Cell Cycle Phases

Cited by 47 publications

References 0 publications

Identification and characterization of CAC1 as a novel CDK2-associated cullin

Identification and characterization of CAC1 as a novel CDK2-associated cullin

The bacterial cytolethal distending toxin (CDT) triggers a G2 cell cycle checkpoint in mammalian cells without preliminary induction of DNA strand breaks

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

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