I did MTT to find the best drug(for cancer) concentration to do Western blot, as drug concentration got higher cell viability got lower, but at the highest concentration has a peak on cell viability. why?
And I compared to my data (2 different range of concentration) that higher range of drug concentration has higher cell viability than the lower range of drug concentration. why?
My first thought is poor solubility with increased conc. A second possibility might be a that the cmpd interacts with the MTT reagent.
MTT assay is a colorimetric assay for assessing cell metabolic activity, specifically mitochondrial activity. It possible that you can see an artifact of your experiment, your drug can interact with the MTT reagent and for this reason, you observed an increase of the mitochondrial activity, not cell viability. I suggest that you do other viability assays, like cell counting marking the cells with trypan blue or check the cell viability by flow cytometry.
Check the media composition while you are exposing your cells to the drug molecule.
The serum present in the media may affect the effectiveness of the drug.
Thomas J Raub and Camila Waltero are right. Some small molecules/drugs do interact with the MTT reagent and can give unusually high values that is independent of cell viability. The other possibility is that your drug is somehow increasing mitochondrial activity and causing this effect; again not an accurate representation of cell viability.
The easiest thing to do will be to look at the cells under a microscope before/after treatment and after MTT addition to see if the number of cells you see correlates with the MTT signal you are measuring. If you see decreasing number of cells with increasing drug concentration but your MTT signal keeps increasing, it tells you that you have interference. Switch to an alternate assay like Neutral red (measures lysosomes), PI/Annexin or one of the release assays like ATP or G6PD or LDH.
Dear Irene, issue with MTT assay is that cell viability (cell growth) determination is done through the intracellular metabolic activity, which in fact is causing the reduction of MTT dye, so it is somewhat an indirect assessment for cell growth or viability. But, what should we keep in mind that the MTT reduction occurs throughout the cell and is affected by a number of factors like changes in cellular oxidoreductase activity, endo or exocytosis flows, intracellular trafficking, test drug or test compound metabolic behavioral deviations and many more. Also make sure readings of your blank and control (100% viable cells) wells are in their appropriate ranges and you dissolved the MTT dye properly. It looks that the highest concentration of your compound here may not be cytocidal due to any of the above mentioned facts.
Regards...
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