Could anyone please tell me the ideal treatment condition for using HDAC inhibitor/ methyl transferase inhibitor on cells? I have 5 azacytidine which is from sigma. The data sheet says that it is highly unstable in aqueous solutions. But most of the research paper states that they treat cells for more than 48hrs. why is it so? If it is unstable, what is the point in treating cells beyond 24 hrs? Does these inhibitors require longer time to carry out their function?
Prepare 5-azacytidine stock solution in DMSO and store @ -20oC. For cell culture, refresh medium containing 5-azacytidine every 24 hours. Treatment schedule may vary depending on cell lines and genes. So you need to perform time-course and dose-response experiments to optimize the treatment schedule.
For TSA treatment, you may start with 500ng/ml for 4 hours. As far as I know, most acetylated proteins including histone proteins have response to this treatment schedule. Longer time will cause cytotoxicity.
For the methylation experiments, inhibition of DNMTs is not enough to remove methylation at CpGs. Cells must undergo DNA replication and cell division in order to eliminate the methyl CpGs that are already present before the addition of 5-aza.
For HDAC inhibitors, you can see effects much more rapidly because histone acetylation is turned over very quickly through the actions of HACs and HDACs. In truth, histone acetylation is not really an epigenetic mechanisms because there is no cellular memory to these marks, even though they are needed for gene expression.
Highly unstable doesnot implicate of it being a immediately broken. Its unsatble in Aq soln, but you can very well use it in DMSO. I think the datasheet should give you that info. Better so, there are ready made Aza's.
Best.
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