I'm planning on exposing epithelial cells to particulate matter, and measuring cell death/viability. I will be using LDH assay on the supernatant, but for a second readout, I would like to use MTT/XTT/MTS/WST-1 to get a readout on a plate reader. All seem to be very chemically similar and work on the same principle of reduction, and all seem to be widely used in the literature, with no obvious reason why some groups use one and other groups use another. I understand that using MTT requires an extra step to solubilise the reaction product before reading, but otherwise, are there any differences? I'm having trouble working out which to use, so any help would be very gratefully received.
I would avoid the redox dependent ones like MTT, pop round if you want a chat
Hi,
as you noted, I generally agree that all approaches are very similar on the biochemical level.
However, the water-soluable derivatives in contrast too good old MTT require an intermediate electron carrier (mostly phenazine metho- or ethosulfate) for efficient reduction of the substrate, which adds another variable to the equation. Still, MTT is also known to be non-specifically metabolized by various other enzymes (e.g. GST) and can be chemically reduced e.g. by sulfur compounds, which may be an issue in certain experiments.
Also worth considering, economically MTT is unrivaled compared to its derivatives, but requires the additional solubilization step before reading the plates. However for most standard research questions, it should not make a substantial difference which particular substrate you use.
MTT is the cheapest one, but you have to dissolve formazan crystals later.
XTT method does not require this step, but you have to mix its solution with PMS before adding it to the cells.
I liked WST-1 the most, but it is more expensive. Also, in XTT and WST-1 methods when you test colorful substances, you have to make additional dilutions of compound without reagent, to remove possible absorbance interference.
And you may find even more methods for testing cell viability: https://www.ncbi.nlm.nih.gov/books/NBK144065/
I like Cell Titer Glo very much. It is quite expensive, but much more accurate.
Be aware of pitfalls of the MTT assay (as well as MTS, XTT, WST-1) resulting in a wrong estimation of viability. Stepanenko AA, Dmitrenko VV. Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability. Gene. 2015 Dec 15;574(2):193-203. doi: 10.1016/j.gene.2015.08.009.
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