With a MTT not actually measuring proliferation, but viability instead. It measures the mitochondrial reductase activity, which represents the 'healthy' state of the cell. It used as a sort of surrogate proliferation assay though, but I would advise you to also consider something like thymidine incorporation or add an apoptosis assay. But for an initial screen MTT is very easy and cheap.

Usually if you test drug effects on cells in vitro the proper characterization of toxic action is to determine the IC50 for each drug on each target cell line. This is the inhibitory concentration that halfs the proliferation of your cells. For this you should perform a 10-fold serial dilution of your drug in the rage of concentration you expect effects and then perform MTS assays. Then you draw a curve by normalizing your results to untreated cells (100%). You will then have the %proliferation function of drug concentration. By extrapolating the 50% proliferation you determine the exact concentration that inhibits cell proliferation by 50%, hence IC50.

There is also the XCELLigence instrument from Roche that measures cell adhesion, spreading and proliferation in real-time and is very usefull for drug mechanism characterisation. It depends on how much you need to invest in this part of your project.

Thnxs to all for your valuable answers. Objective of my study is to use a clinically established drug with wide therapeutic index and less toxicity than most anti neoplastic agents for treatment of cancer. For, this purpose I want to do some in vitro study and if I get good results then I'll do in vivo studies and take it further to trials.

The MTS assay reagent from Promega gives excellent results and does not require the addition of solubilizing agents. I have used both MTT and MTS assays. They are much better than Thymidine uptake in reproducibility and do not require the use of radionuclides.

Hi Prafulla. I think MTT assay measures metabolic activity of the cell, so it is not, by itself, the best reagent for determining cell proliferation. It would be good to add DNA quantification as well.

MTT assay designed to test cytotoxicity of drugs but as it's based on cell metabolisms (succinate dehydrogenase activity) and some of the cells have basic metabolism even without proliferation ability therefore, BrdU which used during DNA synthesis by cells can show better and more trustful results.

Hope it has your response

@John Fassett Thnxs for ur valuable suggestion, but i request u to kindly elaborate DNA Quantification (does u mean to isolate dna and perform its electrophoresis)

I agree with John. If your drug elicits any kind of oxidative response, or damages mitochondria, you may get false readouts. My lab uses almost exclusively fluorescence methods to detect DNA directly. I am particularly fond of the CyQuant kit of Life Technologies. Much easier than BrdU, as it requires no antibodies. Less messy than thymidine incorporation.

To test cell proliferation I would use CyQuant or PicoGreen that quantify DNA content using fluorophores (in line with Guillermo’s suggestion, I would use CyQuant. It is fast, reliable and convenient). Metabolic assays such as MTT, MTS, WST-1 and AlamarBlue may not accurately reflect cellular proliferation rates given that metabolic activity depends on cell growth, growth phase (exponential) and cell cycle and, therefore, does not necessarily correlates with cell number. On the contrary, cellular DNA is independent of metabolic changes. I recommend the lecture of the following paper in which the pros and cons of some of these methods are discussed:

Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research. Quent et al. (2010) J. Cell. Mol. Med. 14(4):1003-1013.