How did you investigate cell death?What do you mena by cells don't look good?

16 micromolar sounds a huge concentration, I would try much lower concentrations and don't forget it's good to use a DNA demethylating agents it's better to check that is working, and cell death it's probably not the best way to go

Hi Michael,

Thanks for your response . I looked at cell death by microscopic observation. When I say the cells don't look good I mean they weren't looking as if they will grow in the AZA containing media well. I will lower concentrations as you said and I will be looking at a positve control gene to male sure that my AZA treatment is working.

What exactly is the aim of your experiment? In case you would like to proof epigenetic regulation of your gene, you would have to look for a cell line that does not express your gene of interest. You could then treat that cell type with low concentrations of 5AZA (5um suffices most of the time) and look at the expression of your gene of interest after the treatment (72hrs should be fine). If you see an expression after the treatment you are confident that the gene might be epigenetically regulated. For validation, you would have to check for CpG island methylation by bisulfite sequencing of your original cell line. Once you find CpG island methylation - you have proven the epigenetic regulation of your gene of interest.In case, you wanted to use 5AZA as a treatment tool - I would also say , that to me the concentrations that you used are higher than clinically applied.

Hope this will be of help.

AzadC 1uM works fine for 293T cells. I observed effect at both, gene expression and methylation level. Be careful with the carrier (most of the times DMSO) because it is also an epigenetic modifier. Good luck!

Hi Wolf, thanks for your response thats extactly what I plan to do.

Hi Jesus, Thanks for your response I dissolve AZA in water.

Hi Pryiya, I can confirm two things that have already been sent as answers to you - first you can use also concentrations as little as 1um - you would have to test that, and second, 5AZA is very unstable - and it might have been destroid once you added the water - you would have to make sure that it is still working by looking at a gene of which you know that it is"off" in your cell line and is methylated.

Good luck, Wolf

Hi Wolf,

I make the stock fresh every time and keep it on ice anduse it on my cells within 10-15min of dissolving. Do you think that much time is OK? Also would DMSO be a better choice then for dissolving AZA?

Priya

Hi Priya,

I usually resuspend 5AZA in the cells media and store single use aliquots, as it is very unstable. I also substitute the media every day, giving the cells a fresh aliquot of drug for 3 days. Hope this helps! Good luck with your experiments!

Sofia

Histone Deacetylase inhibitors (e.g. TSA) are mostly supportive for demethylation (and usually superior to 5-AzaC)

Hi Priya, as already said by many before. Make stock solutions of your 5AZA and freeze these at -80° until use, give new 5AZA to your cells every day of the experiment. In my hands TSA alone or in combination was not superior to 5AZA alone, however this might depend on the gene you look at and at the regulation mechanism that is if histone modification is involved or not. Good luck again. Wolf

According to current view histonen modification precedes DNA methylation

Hi Priya.

I'd advise you to drop doses right down to 500 nM; this is enough to result in reactivation without any direct cytotoxicity. This is in line with what is seen clinically and also on the basis of work I've done using Decitabine (100 nM Decitabine = 500 nM Aza) (Reference http://www.sciencedirect.com/science/article/pii/S1535610812000414 )

As recommended by someone else here, hitting cells with an HDACi is also good for transcriptional reactivation, and if your genes are known to be PRC2 targets you should assay cells immediately after 72 hours, or they will be spontaneously remethylated upon drug withdrawal. (Reference - http://www.ncbi.nlm.nih.gov/pubmed/20631058 )

Hi Ankur,

Thanks for your response.

I will keep these pints in mind

Priya

You need to consider two points carefully: concentration of the drug and duration of treatment. We have used decitabine in Jurkat cells at different concentrations for various time periods up to 72 hrs. We plate cells at low density and treat with decitabine. Since the cells are at low density media need not be changed for at least 3 days. It depends on your gene of interest whether decitabine alone or in combination with a HDAC inhibitor such as TSA will modulate its expression. In this scenario, we treat cells with TSA for 18 hr, wash and resuspend in fresh media and add decitabine and then activate cells to induce gene expression. We prepare stock decitabine and TSA in DMSO and store them in small aliquots at -20o C and avoid repeated freeze-thaw.

Hi

I agree Ankur, you should first determine the effect of drug on lowest possible concentration. Albeit its effect completely depends upon the type of cells you are using. I have used the fresh aliquots of the drug on fibroblast cells and treated for 48 hours and started the conc. with 0.25 micro molar and upto 2 micro molar and it showed variable effects and good study to trace the effect dose dependently.

Hi Sundararajan, Thanks a lot for your response I wanted to know whether adding TSA for 72hrs with AZA is OK or adding at the very end for the last 18hrs is the best way to use HDACi

We pretreat cells with the HDAC inhibitor TSA for 18 hr because its action is rapid. On the other hand, Aza is slow acting and depends on the replication of cells. So, it needs longer exposure to work. Therefore, pretreatment with TSA followed by Aza will be desirable at least in our system. You may want to try both ways and see if either one is better.

As said before, AZA is quite unstable and therefore not very reliable. We have therefore changed to zebularine which gives consistent results. However, there are now publications indicating that AZA and Zebularine may not lead to the same resluts at selected publications.

I think your are using very high concentrations of 5 Aza

I use melanoma cells and effects are observed from low concentrations such as

0.05 to 5 uM.

Can anyone brief me on how the doses are standardized for Azadc treatment and which parameter is considered as a visual marker for the treatment being functional inside the cells?

Do you consider reduced cell viability as a marker for Azadc treatment being functional?