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?
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.
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.
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