I am working in cancer biology. I want to study effect of demethylating agent on expression of certain genes in a cell line. I have chosen the decitabine drug for treatment of the cell line. Which concentration of this drug should I use for MTT assay and what precautions should I take during preparation of drug stock? If is want to give the treatment using 2 drugs at a time, what is the procedure for that- one after the other, or simultaneously?
I think the solubility of decitabine in DMSO is approximately 20 mg/ml. It is used at a final concentration of 1 microM.
Dear Alicja ,
Why you want to use MTT assay in this case ? in the majority of researches applying flow-cytometry to measuring effects of drug in cancer cell line....
But i think you should use 3 or 4 different concentrations of this drug like : 0.5 μg/ml, 5 μg/ml, and 20 μg/ml decitabine for 72 h.
It is a suggestion, try to use flow-cytometry if you want to measure apoptosis effects of this drug. ( Annexin-V and PI double staining ).
Dear Madhuram,
Why you want to use MTT assay in this case ? in the majority of researches applying flow-cytometry to measuring effects of drug in cancer cell line....
But i think you should use 3 or 4 different concentrations of this drug like : 0.5 μg/ml, 5 μg/ml, and 20 μg/ml decitabine for 72 h.
It is a suggestion, try to use flow-cytometry if you want to measure apoptosis effects of this drug. ( Annexin-V and PI double staining ).
While I don't know too much about it, I did recently write a related article for EpiGenie covering a pretty cool paper on the topic . Hopefully this will help http://epigenie.com/decitabine-scores-a-cancer-therapy-hat-trick/
Have you looked if InSilico DB has public datasets of interest (your Cell line and drug of interest) I know that the CMAP I and II are available.
Once you identify your datasets you can export them to different analysis tools in a ready-to-analyze format. Tools include Gene Pattern, Gene-E, IGV, Ingenuity iReport.
You can get from a dataset to a heatmap that shows over-expressed genes in one condition VS another in minutes (tutorial: https://insilicodb.org/save-time-1-click-high-quality-annotations-and-genomic-data-in-gene-e/).
You can also create new datasets by combining samples from various public datasets.
It's publlshed in Genome Biology and is accessible here: https://insilicodb.org
Hope this helps
Dear Hamidreza Aboulkheyr Estarabadi
Thanks for ur valuable suggestion but MTT assay will also tell about the toxicity or viability effect of that drug on cell line and my concern is to get a optimum dose for further experiment. so what is benefit to use flowcytometer for determination of optimum conc. of dose for further experiment or you tell in what way use of flowcytometer will be superior in stead of MTT assay.
Dear Madhuram Khandelwal,
Thank you for your feedback. ok. it depends on the types of result that you want. Qualitative or Quantitative which one. When you want to know about which dosage is optimum, i think it means that how many of cancer cells are died and how many of them are still alive rite? So in this case, you can analyse both of them at the same time by Flow cytometry ( Qualitative and Quantitative ( # of cells )). So based on the obtained results from Flow-Cytometry in different concentration of drug you can analyse and fine optimum dosage ( Based on the % and # of death cells ).
Dear Madhuram Khandelwal
in this paper, flow-cytometry is used for optimum dosage of drug ( Decitabine )
http://www.sciencedirect.com/science/article/pii/S1382668911001323
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