Hello, Abdul!

The MTT test is used to assess cell viability by measuring the activity of mitochondrial dehydrogenases. Since viability is the reciprocal of cell death, MTT can be used to determine cell death, mainly when cell death is associated with impaired mitochondrial function (internal pathway of apoptosis, cytochrome c release, activation of caspase-3, caspase-9).

Hello Abdul

You are right. MTT is not the assay to be used to assess cytotoxicity. There are many limitations as far as MTT is concerned.

The rate of tetrazolium reduction could be because of general metabolic activity. The rate of MTT reduction could also change with pH as well as the concentration of glucose in the medium.

Addition of solvents to solubilize formazan crystals could be damaging to the cell architecture and enzyme activity.

MTT reduction can take place even outside the cell at the plasma membrane or even in the cytoplasm. So MTT reduction is not just related to the mitochondria. These drawbacks make MTT as not the right choice for cytotoxicity assay.

Thank you.

MTT is not the best option but because it is easy to preform and cheap it is widely used. I usually do multiple methods to evaluate cytotoxicity starting with MTT, then PI staining and flow cytometry and then western blot for cleaved caspase 3, caspase 9 and PARP.

MTT tells me how many cells are viable (metabolically active compared to untreated control). If less, it points to less cell proliferation, cell death or less metabolic activity. PI staining allows me to asses progression of cells through cell cycle and points out the cells with less DNA amount (apoptotic ones). With western I prove if the apoptosis is canonical through Caspase cascade or if not I try to identify another signaling pathway leading to cell death (autophagy, necrosis, mitophagy...).

Hi!

I agree with the previous answers, the MTT assay is performed to assess cell viability. It measures the reduction of a tetrazolium component (MTT) in an insoluble formazan product by the mitochondria of viable cells, from a yellow water-soluble tetrazolium dye, to purple formazan crystals caused mainly by mitochondrial dehydrogenases. The MTT assay measures the growth rate of cells in a linear relationship between cell activity and absorbance.

Hi Abdullah Abdullah ,

I agree with you and previous commenters that MTT, SBT and Crystal Violet assays are "Cell Enumeration" assays and not "Cell Viability" assays. They serve as a proxy to estimate the derivative number of living cells in comparison to an "untreated" control but do not actually measure the number of viable, apoptotic and necrotic cells.

One perfect solution to this problem is the Apo-Tox-Glo Triplex assay (Promega/Fisher) which measures both viability and apoptosis as well as necrosis/cytotoxicity.

See: https://worldwide.promega.com/products/cell-health-assays/apoptosis-assays/apotox_glo-triplex-assay/?catNum=G6320

It is much more sensitive, all in one well, and highly reproducible. It's always better to use something that directly measures the outcome rather than inferring it from a proxy/surrogate estimate.