Are you sure the cells are dead? Depending upon a cells metabolic activity MTT is converted to its coloured formazan product. This process is toxic to the cells but the amount of formazan produced indicates the cells metabolic activity independent of this. If you solubilize the formazan do you measure absorbance in the "dead" cells? The amount of absorbance could be low if the cells are not thriving prior to the addition of MTT.

In my opinion, from the image above, it looks like you used too much force when pipetting and cells started to detach. Try to do it more slowly.

Hi, I'm finishing up my PhD in a venom analysis lab. I do massive amounts of toxicity, cytotoxicity, toxic mechanisms, differential and selective toxicity, and use everything from counting cells by eye, by trypan blue stain using a Countess, by various flow cytometric assays, and my PI has been at the forefront of venom analysis for decades. When I am contracted by pharmaceutical companies trying to develop a drug they prefer MTT assays as their initial "cytotoxic or not" and "approximate EC50 dose". Even though it's indirect it is far easier to run multiple replicates of multiple doses even when you have little material (such as an isolated scorpion venom protein... it takes a lot of, potentially hazardous, work to get a few micrograms sometimes). So I am intimately familiar with MTT and (if you haven't looked into this, I recommend it) MTS.

As for why you are seeing cells die after the addition of even small amounts of MTT it is because MTT is extremely cytotoxic to any cell that produces NADH (so... cells that do cellular respiration) is that MTT kills cells. "Intracellular metabolism of MTT has been shown to gradually cause mitochondrial injury, disturbance of normal cell metabolism, and finally cell apoptosis [4,41]. Time-dependent loss of membrane integrity as a result of formazan exocytosis has also been proposed as a mechanism of cell death following MTT incubation" from Article The MTT Assay: Utility, Limitations, Pitfalls, and Interpret...

I don't think you pipetted too hard. You need to titurate cells well following MTT treatment to get a good result and you need to solubilize the cells with DMSO or another detergent. Formazan (reduced MTT) forms insoluble crystals, you can see these after treatment with MTT, these have been shown to build up in multiple organelles including mitochondria, ribosomes, nucleus, and not only interfere with cellular operations but can pierce and lyse cellular membranes in a similar manner to any crystal forming within a cell (such as ice). Even in extremely low doses I think you would witness apoptosis induction in healthy cells, and the loss of adhesion due to loss of membrane integrity and effective protein synthesis and the consumption of the NADH necessary to maintain vaiability. MTT is not an assay that is meant to result in intact and definitely not living cells. It is essentially quantifying the amount of reducing agent present (which is typically correlated to cell number, but it varies for every cell type and every treatment and you should always have an identical treatment ladder with varying cell numbers ranging from zero to double what your treatment wells will be seeded with). The nice part of this is that I have found that incubating my cells with MTT overnight results in a far more consistent result, and the use of a ladder as described below demonstrates whether the cell numbers used, incubation time, MTT dose, and carrier provided a relationship between seeded number of cells and optical absorbance that has (for my purposes) an R2 value of over .9 for either logarithmic, asymptotic, or linear best fit lines. It's also acceptable, in my opinion, to exclude the double population and lowest population if these are outside a clearly linear, with high R2 value, relationship for all other cell numbers and absorbances. However, this also means that you should not be including any treatments where the results were outside your new maximum and minimum (the lowest viability would be the lowest population you included in you best fit defining data from the ladder). This is also typically only useful in the case of analyzing cytotoxins by dose and trying to find the dose that results in an equal absorbance as if you simply seeded the wells with half the cells, since if you have 100%, 50%, 25% and 12.5 % but 200% and 6.125% were not within the linear range it doesn't mean you need to throw out all your data... most treatments will be somewhere between 100% and 12.5% relative viability anyway... assuming there was some time that the cells were alive and produced some NADH then even only 1 hour should have been eough time for the 100% population to produce as much as a population 1/24th it's size if incubated for 1 day.

Anyway, I just wanted to elaborate a bit and explain why I am of the opinion that trying to keep your cells alive after exposure to MTT is an unlikely task. MTS is water soluble and does not form crystals and it may be possible to find a dose that is both observable colorimetrically and may not be completely wiping out the populations... but for MTT I don't think you would get surviving cells if you've added a dose that is useful for quantifying things at any level.

. If you don't use a ladder (every single plate i use for an MTT assay has 4 columns devoted to a ladder, the first containing double the cell population as my treatment wells, the second identical, the third half my treatment, then a quarter, then an eighth, and finally a media only and a carrier +media only, this ladder should result (with 4 reps at each population) in relatively consistent results (they should be seeded at the same time as the rest of your wells and treated identically to your control wells, same volume of MTT, same incubation, read at the same time... it's on the same plate so this shouldn't be difficult to make the conditions close to identical except for position on the plate... so remember that corners and edges evaporate faster and will often seem slightly darker and more absorbant as the same quantity of f

Randall Scott Gieni

Thank you for your answer

regarding the absorbance, yes I measured the absorbance after 2 and also 4 hours, and the absorbance decrease after 4 hours, and by observation under microscope, I saw a lot of aggregated cells which confirmed that exposing the cells with MTT assay for longer time, make them go to the dead phase!

Winda Ariyani

Thank you for your answer

I repeated the experiment again with more gentle addition of MTT but no difference, we had the same problem like the last two times. We had some precipitation of MTT and having the abnormal cell morphology.

Tanner Harvey

Thank you for your well explained answer

As you told we saw some precipitation of formazan which is not favorable!, I just replaced the assay of proliferation with another method (using presto blue), which the result was quite conclusive.