Hi everyone,
Has anyone experienced ambiguous or conflicting results between WST-8 and MTT assays in cytotoxicity studies?
I am currently testing two peptide candidates (SKP85 and SKP179) for potential cytotoxicity on HaCat and HDF cell lines, using concentrations ranging from 20 µM to 0.625 µM. I performed both WST-8 and MTT assays.
For the WST-8 assay, I observed >90% cell viability at all concentrations for both peptides after 24 hours in both cell lines. However, with the MTT assay, SKP179 at the highest concentration showed about ~50% viability after 24 hours, while concentrations of 10 µM and below gave results consistent with the WST-8 assay (>90% viability). I did not observe this discrepancy for SKP85. Both assays were repeated with 3 biological replicates and showed consistent results within each assay.
Has anyone encountered this before? What could be causing this difference?
A quick search shows that MTT itself can be toxic at higher concentrations and depending of incubation time and cell type.
Hello Alif Ismail
There are a few differences between these two assays. Perhaps these differences could be the cause of conflicting results between them.
a. In MTT assay, a yellow tetrazolium salt, is reduced to purple formazan crystals by mitochondrial dehydrogenase in living cells, while in WST-8, it is the cellular dehydrogenases.
b. In MTT assay, formazan crystals are insoluble in water and requires a solubilization step with solvent like DMSO, while in WST-8, the formazan produced by WST-8 is soluble in water, eliminating the need for a solubilization step.
c. As mentioned by Dr. Patricia V Pietrantonio, MTT compound is cytotoxic and only remains stable for a few hours, while WST-8 has a much lower cytotoxicity and is stable for up to 48 hours.
d. WST-8 offers a wider linear range than MTT assay, allowing for more accurate quantification over a broader range of inputs. This wider linear range can be particularly useful in experiments where cell populations or concentrations may vary significantly, or when analyzing samples with high variability.
e. WST-8 assay has a higher sensitivity than MTT assay.
Best,
WST is more sensitive than MTT, I would recommend you try a third viability assay to make a firm conclusion like the Muse Cell count and viability assay. I have heard it can provide more accurate results
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