I want to study protein localization during cell division and before cytokinesis. So, for that I want to synchronize the cells. Please suggest how I can achieve this.
Hello,
there are several methods, each one with different pros and contras... I suggest you to have a look at this recent Nature Protocols paper, Rosner et al. Nat Protocols 2013 (doi:10.1038/nprot.2013.011).
For a quick look you have a comprehensive table is at this link: http://www.nature.com/nprot/journal/v8/n3/fig_tab/nprot.2013.011_T1.html
Hello,
there are several methods, each one with different pros and contras... I suggest you to have a look at this recent Nature Protocols paper, Rosner et al. Nat Protocols 2013 (doi:10.1038/nprot.2013.011).
For a quick look you have a comprehensive table is at this link: http://www.nature.com/nprot/journal/v8/n3/fig_tab/nprot.2013.011_T1.html
Within the link provided by Pietro, they list Nocodazole. We have used this in our lab several times, and it seems to work well for halting cell cycle just before mitosis as MTs are needed for any further separation of the chromosomes. For an example protocol, see http://www.scienceboard.net/resources/protocols.asp?action=article&protocol_id=125
A good process for synchronization of cells is to use an excess thymidine block, which results in block in DNA synthesis (well feedback inhibition on the production of dCTP, which prevents DNA synthesis). For the best results, try a double round of thymidine blocking, with ~2mM thymidine for 24 hours, 12-16 hours without thymidine in media, and then another ~18-24 hours in thymidine again. Upon the last washout, all the cells should begin to enter S phase. These conditions should be optimized for your cell lines. A DNA dye may be useful to count cells in each phase while you optimize.
*edit: here is a paper where a former graduate student in the lab performed this cell synchronization assay. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1783781/
The simplest method, as already mentioned, is serum deprivation for 24 to 48 h. It is important to assure that certain basic requirements are met to maintain viability; e.g. we include selenium (10 nM) and transferrin (5 ug/ml) in the culture media. Low serum (0.1%) may work better than no serum at all if your cells are particularly sensitive to stress.
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