In my experiment I got false MTT activity in-spite drug was supposed to be cytotoxic I just want to get insight about limitations of MTT.
As a background control, lyse some wells with detergent to determine MTT reduction caused by cytoskeleton/extracellular matrix components.
Drugs that interfere with mitochondrial respiratory chain can yield to over- or unerstimations of cell survival.
More, if your compound has redox properties, it may directly react with MTT or formazan. The most common case is the presence of antioxidants in herbal extracts. Washing the cells prior to adding the MTT solution can limit this unwanted reaction.
In our lab, we suggest performing a second survival assay which is not dependent upon metabolic activity (eg. crystal violet, protein quantification,...). This will allow you to check for any discrepancy with your MTT.
Some labs use MTS a supposed imprivement. I use neutral red (Nature 2008)
What type of error you got. If say that we can troubleshoot. Usually we won't get such false activity. Be sure regarding number of cells while seeding the plates.
Valerian is correct - MTT is actually measuring NADH levels, so anything that affects mitochondrial respiration will affect the MTT signal. Second opinion is a must if the MTT results are in doubt, and there are plenty of choices out there - from DNA dyes to LDH kits. That will hopefully resolve your mystery. Good luck!
You can see nice comparison of various assays in this review. http://www.nature.com/nrd/journal/v10/n3/full/nrd3373.html
Some of basic recommendations its to clean cells by PBS to eliminate major background; then make a none posed group for comparison and a isotype positive to dead like IP or another kind of substance to induce to dead all cells.
And maybe if you need to compare the stage of apoptosis you will use Anexina V/ PI.
Another point of view its the redox stat of your sample to chose other method of viability.
sometime, after 2 hs of incubations, cells died for crystals of mtt.
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