期刊
GENES
卷 13, 期 3, 页码 -出版社
MDPI
DOI: 10.3390/genes13030408
关键词
DNA replication; S phase; EdU-BrdU pulse chase; cytometry; cell lines
资金
- Ministere de l'Enseignement Superieur et de la Recherche (MESR)
- Ligue Nationale contre le Cancer
- Association pour la recherche sur le Cancer (ARC)
- SIRIC (Site de Recherche Integree sur le Cancer) Montpellier Cancer
- Association pour la Recherche sur le Cancer (ARC) [5101]
- Institut National du Cancer (INCa) [PL110]
This article describes a simple and robust method to measure the duration of the S phase in cell cultures. It utilizes a dual EdU-BrdU pulse-labeling regimen with incremental thymidine chases and quantification by flow cytometry. The method does not require cell synchronization or genome engineering and can be used for various types of cells.
Eukaryotes duplicate their chromosomes during the cell cycle S phase using thousands of initiation sites, tunable fork speed and megabase-long spatio-temporal replication programs. The duration of S phase is fairly constant within a given cell type, but remarkably plastic during development, cell differentiation or various stresses. Characterizing the dynamics of S phase is important as replication defects are associated with genome instability, cancer and ageing. Methods to measure S-phase duration are so far indirect, and rely on mathematical modelling or require cell synchronization. We describe here a simple and robust method to measure S-phase duration in cell cultures using a dual EdU-BrdU pulse-labeling regimen with incremental thymidine chases, and quantification by flow cytometry of cells entering and exiting S phase. Importantly, the method requires neither cell synchronization nor genome engineering, thus avoiding possible artifacts. It measures the duration of unperturbed S phases, but also the effect of drugs or mutations on it. We show that this method can be used for both adherent and suspension cells, cell lines and primary cells of different types from human, mouse and Drosophila. Interestingly, the method revealed that several commonly-used cancer cell lines have a longer S phase compared to untransformed cells.
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