Double Thymidine block is used to synchronize cells. With this system it is possible to obtain cells arrested in the G1 / S phase that will follow cell cycle in a synchronized fashion for more or less 2 divisions. My question is, how long is it possible to keep this G1 / S arrest? I would ideally like to keep them likewise for at least 24h. The ultimate purpose is to do a siRNA KO and study a protein which is essential in M phase. Keeping this KO over a period of 24h would be really deleterious for the cell population thus I have to prevent mitosis to happen and thought 2x thymidine could be used without doing the second release for 24 hours after the point in which it is supposed to do the release. If anyone knows a better way to solve this it would be great.
Hello Iñigo Lobregat !
It should be noted that synchronization of
cells by “thymidine block” influences not only DNA
replication but also RNA synthesis [Kasten, F. H., Strasser, F. F., and Turner, M. (1965) Nature(London), 207, 161-164]. So it is not recommended to incubate cells with high thymidine content for more than 16 h [Stein, G. S., Stein, J. L., Lian, J. B., Last, T. J., Owen, T. A., and McCabe, L. (1998) in Cell Biology: a Laboratory
Handbook, 2nd Edn., Vol. 1, Academic Press, N. Y., pp.
253-260].
More information about different methods of cell synchronization you can find in my review:
Rustem E Uzbekov
Analysis of the cell cycle and a method employing synchronized cells for study of protein expression at various stages of the cell cycle
Biochemistry (Moscow) 06/2004; 69(5):485-96. DOI: 10.
Full text you can find on my ResearchGate page
Best wishes
Rustem Uzbekov
alright I was not aware of that and it is indeed useful information, thanks!
Most knockdowns in cell lines are maximal somewhere between 24-72h. Once you know the kinetics of your specific RNAi-mediated depletion (increasing mitotic index), you can time your S-phase arrest accordingly, no need to arrest before. Is the goal to study your protein in mitosis or outside of mitosis?
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