Dear Juma Almutaani

The MTT assay relies on the reduction of MTT to a colored formazan product by mitochondrial enzymes in metabolically active cells, which is true. While this reduction is generally associated with viable cells, it can also occur to some extent in cells undergoing apoptosis or necrosis, particularly if they have not yet completely lost their metabolic function.

The MTT assay can sometimes produce false-positive results, indicating more viable cells than are present, especially when cells are exposed to certain compounds that interfere with mitochondrial activity, like certain drugs or nanoparticles, which can also affect the assay's accuracy. Please note that dying cells can still exhibit some level of MTT reduction.

So, while the MTT assay is a widely used tool for assessing cell viability, its limitations in differentiating between live and dead cells necessitate the use of complementary assays to obtain a more comprehensive understanding of cell health and death.

There are other assays that can be performed which can differentiate between live and dead cells such as:

1. The Trypan Blue Exclusion assay which relies on the principle that viable cells with intact membranes exclude the dye, while dead cells take it up. It is a simple, visual method using a microscope.

2. Assays using fluorescent dyes like Annexin V and propidium iodide (PI) can differentiate between early apoptotic, late apoptotic, and necrotic cells, providing a more nuanced view of cell death.

Regards,

Malcolm Nobre

Thank you Malcolom Nobre for valuable information.

Hi !

The MTT assay differentiates live from dead cells based on the metabolic activity of mitochondrial enzymes, specifically succinate dehydrogenase. Viable cells with intact and functional mitochondria reduce the yellow tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to an insoluble purple formazan product via NAD(P)H-dependent oxidoreductase enzymes. This reduction occurs only in metabolically active (i.e., live) cells, as dead cells lack the enzymatic activity required for this conversion. The resulting purple formazan crystals are solubilized using an organic solvent like DMSO or isopropanol, and the absorbance is quantitatively measured at 570 nm using a spectrophotometer, providing a direct correlation with the number of viable cells.

Thanks for the generous information, Dr. Mani Sharma. Well done.

The MTT assay distinguishes live from dead cells by measuring mitochondrial activity in viable cells. The process relies on the reduction of the yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium salt into purple formazan crystals by active mitochondrial enzymes, primarily succinate dehydrogenase, in metabolically competent cells. Only live cells with functional mitochondria can perform this reduction, while dead or metabolically inactive cells lack this capability, resulting in no formazan production. After incubating cells with MTT for 2–4 hours, the insoluble formazan crystals are dissolved using a solvent like DMSO or isopropanol, and the absorbance is measured at 570 nm (with a reference wavelength around 630 nm to account for background). The absorbance intensity correlates directly with the number of viable cells, as higher values indicate greater metabolic activity and cell viability, whereas reduced absorbance suggests cell death or toxicity. However, the MTT assay does not directly detect dead cells and may be influenced by factors such as metabolic inhibitors or over-confluent cultures. For comprehensive viability assessment, it can be combined with other methods like LDH release assays that specifically measure cell death. Let me know if you need further details on protocol optimization or alternative viability assays.

The MTT assay effectively differentiates live from dead cells by detecting mitochondrial activity, a key biomarker of cell viability. This colorimetric method relies on the ability of metabolically active cells to reduce the yellow MTT tetrazolium salt into purple formazan crystals through mitochondrial enzymes, particularly succinate dehydrogenase in the electron transport chain. When MTT is added to cell culture, viable cells with functional mitochondria actively take up the compound and convert it to insoluble formazan precipitates, while dead cells lacking metabolic activity fail to produce this conversion. After a typical incubation period of 2-4 hours, the formazan crystals are dissolved using solvents like DMSO or isopropanol, and the resulting purple solution's absorbance is measured at 570 nm, with intensity directly correlating to the number of viable cells. The assay's specificity for mitochondrial function makes it particularly valuable for assessing cell viability, though it's important to note that it measures metabolic activity rather than membrane integrity, which can sometimes lead to discrepancies - for instance, quiescent cells with low metabolic rates may show reduced signals despite being viable, while certain cytotoxic agents affecting mitochondria might produce false positive signals. Additionally, the MTT assay cannot directly quantify dead cells and may require complementary methods like LDH release assays for comprehensive viability assessment. Factors such as cell confluence, incubation time, and potential formazan crystal aggregation must be carefully optimized to ensure accurate results. Despite these limitations, the MTT assay remains widely used due to its simplicity, cost-effectiveness, and reliable correlation with cell viability across most mammalian cell types, making it particularly useful for drug screening, cytotoxicity tests, and proliferation studies where relative metabolic activity serves as a valid proxy for cell health.