I was doing a MTT assay on 2DG for my PC3 and LNCaP cell lines for my project, however I am not sure why MTT is used. (I mean due to time restriction of the project, I did MTT to save time but what are the disadvantage for using MTT? Since I read from most paper MTT is not the best approach for dealing with cytotoxicity, but instead for measuring viability or metabolic activity of the mt)
You may find the answer to your question here https://www.researchgate.net/post/Anyone_familiar_with_LDH_assay_and_MTT_assay
Hi Conrad,
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is commonly used to determine whether cells are viable or non viable. The metabolic activity of viable cells has the ability to reduce the tetrazolium dye to its insoluable form which is formazan; purple crystals and then we can measure the absorbance which will allow us to identify the amount of viable cells.
However, MTT can also be used to quantify the cytotoxic index in cell populations. Attached is an article where the MTT assay was successfully used on cancer cells that had been treated with different chemotherapeutic drugs.
Article Comparison of Cytotoxic Activity of Anticancer Drugs against...
The difference when comparing between cytotoxity and viability is that when you use the MTT assay for viability you are determining the proliferative index of the cells themselves in most cases without the addition of any treatment; you are looking for an increase in absorbance as the cells should be reducing the tetrazolium dye and forming formazan which you can calculate through an absorption reading.
With the addition of a cytotoxin you will be looking at the decreasing absorbance of the cells that have been treated with the drug. A decrease in the absorbance of the cells treated with increasing concentrations of the drug in comparison to the control cells which have not been treated by the drug will suggest that there is cytotoxicity.
In term of the disadvantages associated with the use of the MTT assay on cytotoxicity. Another article has been attached which contains a table (1) which states the disadvantages associated with the assay. Further references are also present in the article if required.
Article Limitations of the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphen...
Hope this helps
MTT requires a functioning mitochondria to measure the metabolic activity of live cells. However, some cells can be perfectly viable but not necessarily with a lot of metabolic activity. Also it is time dependent and you need to ensure that you are measuring activity at the most active part of the cells growth phase. Accordingly you must validate the results by directly measuring cell apoptosis. If your cell are attached you can try SRB assay.
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