Establish the IC50 dose for your cell line (check the literature or calculate it yourself if not previously reported). I'd suggest a single dose (IC50 dose) at time zero, then take RNA, DNA and measurements of viability, etc. at time points of up to about 120 hours after treating your cells (comparing with vehicle-treated controls), but this would depend on the particular cell line you're using.

I agree with Matthias and would like to add that demethylation is achieved through DNA repair due to the covalent attachment of any of the DNMT (not only DNMT1). I think a quick way would be to perform quantification of global DNA methylation with and without 5-aza using the antibody against 5 Me-C. You can either perform an ELISA, slot or dot blot with total genomic DNA. If you don’t have a slot or dot blot vacuum manifold you can do it manually by spotting 1µl (1-10 ng) heat-denatured DNA on a nitrocellulose membrane and proceed as if it were a western blot. I use to use Hybond membrane and I also UV-crosslinking but I think people don’t do it this way.

Be careful with this stuff its unstable in aqueous solutions.

I agree with Matthias Schaefer. Don't forget to renew the medium every day due to aza poor stability.

It is especially important to make up your azacytidine or azadeoxycytidine in ice cold (0 to 4C) water or buffer and to do all your dilutions and filtrations at 0 to 4C. I recall that sometimes 5-azadeoxycytidine didn't dissolve well in ice cold water or ice cold PBS but after storing in the undissolved 5-azadeoxycytidine and water or buffer mixture in the freezer over the weekend it dissolved after thawing on ice. Ice cold includes the syringe and sterile filter you might use to sterile filter it. Store your solutions at -20C (or colder). Thaw on ice. We only raised the temperature in the very last step when we made a dilution into warmed medium to treat the cells (less than one minute used for this step).

We used Millipore SLGV004SL filter units to filter sterilize 5-azadeoxycytidine and other drug solutions in our studies. These have a low volume and low surface area. We would rinse these with buffer (and combine the rinse with the filtered drug solution) to recover some drug that might have bound to the filter.

I am not sure that after Aza treatment one should assay for transcriptional reactivation of a gene or genes to asses drug-mediated DNA demethylation unless it has already been reported. Still though, one will need to perform DNA methylation analysis either globally or on the loci he is interested in. For some genes, DNA demethylation is not a sufficient trigger for their activation and if it does, its effect may be very weak. It is well known that there are subsets of genes which need also a synergetic action of HDACs inhibitors for appreciable reactivation.

Also, I think the duration of the treatment has to do also with the duration of cell cycle for the cell line being considered. Because of the mechanistic action of the 5-Aza drug, one needs several rounds of divisions to make real difference in the pattern of global DNA methylation. I have been told also that there are side effects for which it would be appreciated if people want to share their experiences. So, the dose should be kept at its minimum.

Culture your cells with 10 μmole/L 5-Aza-2′-deoxycytidine (5-Aza; Sigma–Aldrich, St. Louis, MO) for 7 days. The culture medium should be changed every days. At the end of the experiment, you can harvest the cells for cell death assay, intracellular ROS production, bisulfite genomic DNA sequencing, real-time RT-PCR and western blotting. 5-Aza is typically dissolved at 50 mg/ml in 50% acetic acid to yield a clear colorless solution. 5-Aza is also soluble at 50 mg/ml in DMSO or 0.25 mg/ml in water.

I would like to point out that in my experience different cell lines vary quite a lot in terms of toxicity to this compound, as well as their response in demethylation. Also one should take into consideration that because the compound does not actively demethylate the DNA, but rather inhibits the methylation of new DNA, then the dose response should also be done for a relatively long period of time. The more cell cycles you can get through, the less methylated your DNA should be.

I agree Matthias and Matthew. You have to test the cytotoxicity on your cells but, usually, we 're working with 1-10µM during 72 hours. Cells have to be treated during at least 1 double time. Then if the double time of your culture cell is 24 h, you have to treat 24 or 48 heures. This drug is unstable in aqueous solutions, then you have to change the medium and add new 5azadC every day.

Just add one more tip. In my case I can only make my experiment work at 25uM. So you might want to add a higher conc to test your sample.

Yes I'm in agreement with Yunkun Dang, you should also add a higher conc. treatment. I had used 0.25 uM to 5 uM concentrations of Azacytidine on Fibroblast cells and results were interesting on higher doses. Although I haven't experimented my cells on higher concentrations but it is a good option to check any interesting changes on higher concentrations and you should also know the doubling time of your cells.. All the best..

Does anybody has an idea, if RNU6B is influenced by 5-Aza?

Yea! Stability is the biggest issue with 5-Aza drugs. I had experienced this very well. However, one should not used this drug for more than 1-2 weeks, when dossolved in medium. In my experiments, I always prefer to use freshly (around 1-1.5 week), then I don't have any stability issue. Also, just to add a note, if you use Aza from Sigma, you can dissolved/reconstitute it in DMSO, which is more stable at around 4 degree (atleast in my hand).

The expression assays should be done both in the presence and absence of the drug. This is because remethylation and reversion of silenced state may occur in some loci.

Palsamy Periyasamy, Does 5-aza dissolved in DMSo turn solid (like ice) when you keep it in 4 C degree?

What would be a good/ideal stock solution for 5-aza deoxycytidine ? I am thinking to make 10 mM stock in DMSO and aliquot 50 ul. Also do anyone know if the solution can be refrozen and reused or every time we need to thaw a new aliquot.

Also when we talk about 1-10 uMole conc., is it final concentration in medium?

I would not recommend storage of 5-azaC at 4C degees because hydrolytic pocesses (ring-opening reaction to the formyl derivatives of deoxyribofuranosylcarbamoylguanidines) may continue even at that temperature. Keeping the 10mM 5azaC stock in 50% DMSO in the freezer is probably the best.way to keep the drug active for a longer period.

The final concentration in medium should be empirically determined for particular cell line and experiment. This is becasue the hypomethylating effect and toxicity is cell line dependent. In most cases, 0.1-5 micromol/L 5azaC is the final concentatrion in the medium. For plant cultures concentration should be increased up to 50 micrmol/L.

 can we store the sealed powder form at room temperature?

is 5-azacitidine is soluble in DMEM , so i can dissolve it in the media just before adding it to the cells?