It was commonly accepted in during my student years in the lab I was working, that when you add the MTT reagent (which is toxic to the cells), the cells die.
So my question is if they do, how long are the dead cells still able to reduce the MTT reagent, and what exactly is the point of the incubation with the reagent if the dead cells are not able to reduce MTT anymore?
On the other hand, the MTS is not supposed to harm the cells, so if some external factors influence the cell viability during the 3h incubation with the MTS reagent it could influence the reading.
So basically I would like to know what is happening with the cells during the incubation period with the MTT and MTS reagent. Anyone here to explain?
Dear Alja Štern
There are some good points that you have mentioned. So, what happens to the cells during the incubation period with MTT/MTS reagent?
MTT reagent exhibits cytotoxic effects and adding the reagent to estimate cell viability may actually be damaging or even killing cells during the incubation period. Exposure of cells to MTT may result in dramatic changes in morphology during the formation of formazan crystals. The MTT assay uses reducing equivalents such as the co-enzyme NADH to convert MTT into a colored formazan product. Diverting NADH away from critical cellular functions and towards reduction of MTT is likely to have adverse effects on cell health. Also, formazan crystals themselves may cause damage to membranes during exocytosis.
Although the MTT assay results generally correlate with the number of viable cells growing in standard culture conditions, it is not so! The rate of tetrazolium reduction reflects the general metabolic activity. The rate of MTT reduction can also change with culture conditions (e.g., pH and glucose content of medium) and the physiological state of the cells. Additionally, cells growing rapidly as a monolayer will have different rate of metabolism than those that have undergone differentiation, grown into a confluent monolayer or have become senescent.
I agree that MTS is not supposed to harm the cells, but a large proportion of the reading obtained in commonly used culture media in MTS assay could be caused by media component amplification of formazan production in a way that cannot be corrected for by media-only controls. Also, the albumin present in 10% serum could reduce the assay absorbance.
So, cell conditions during the incubation period with the reagents does influence the readings in MTT/MTS assay.
Best Wishes.
Dear Malcolm Nobre , thank you very much for your answer.
I was also worrying about the serum or possible other components including the tested substances to influence MTT reduction.
Would you recommend to discard the media before MTT addition? Some do not recommend it due to the possibility of some cell loss during the process, but I think the possibility of the media influencing the reading is far more concerning.
For MTS I am not sure because you need sufficient volume for the readings. So discarding the growth media, and adding (media without serum, or PBS) and than MTS?
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