I did an MTT assay on HT-22 cell line of my drug. I got the higher reading in my treatment sample, it shows that the drug is proliferating the cells, because the reading becomes higher, when I increase the time of treatment.
I want to ask, how I should show it in the bar diagram, or should I go for the other cell viability assay to confirm?
Ok - a bit more info required but I think I know what you are getting at.... You do need untreated cells to normalise to [your drug at X concentration, to get a % inhibition] for each time point i.e. you cannot compare treated versus untreated cells on different days [if that is what you are doing] unless you want to show relative proliferation rates over time? But it does sound like your drug isn't doing much, at least in a short term assay - you may consider prolonging your drug treatment to 7-14 days (i.e. low density seeding, replenishing drug/media every 2-3 days) and quantify by colony formation or some other method - and normalise to untreated cells (i.e. DMSO only) against an increasing dose of drug. It is, of course, quite possible that your drug really does increase proliferation of your HT-22s - which could raise some interesting biological questions for you!
You should perform tests for both time dependent effect and dose dependent effect of the drug on your cells. You also will need to assess if:
1.Drug is degrading quickly in the media? (then you will see no effect or may actually promote cell proliferation)
2. the drug getting modified by the cells?
3. The cells are actually able to internalize and/or metabolize (modify) the drug?
Another method for cell viability- Staining the cells with Tryypan blue and count the dead and live cells (cell counter)
MTT actually measures metabolic (mitochondrial) activity, not proliferation directly. Perhaps your drug is altering the metabolism of your indicator cells. I agree that a dose response and a time course are good ways to determine if your drug is actually increasing proliferation or inhibiting apoptosis. Checking the viability by trypan blue is definitely worth doing.
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