I was performing an MTT assay to check cell viability. Due to a technical issue in the plate reader I was using, I had to read the plate at 540 nm wavelength. Would the results still be credible?
Dear Ksh,
The MTT assay results at 540 nm can still be credible but may underate cell viability compared to the standard 570 nm reading. However, the deviation should be noted, and the data interpreted with caution. I will suggest validating against standard readings if possible.
Hello Bahar Ksh
If you have to read the plate at 540 nm wavelength, use DMSO as the solubilizing solution.
If you are using acidic isopropanol as the solubilizing solvent, the absorption maximum is at 570 nm. But for DMSO as the solubilizing agent, the absorption maximum shifts to 540 nm.
Best.
Bahar Ksh
The MTT assay typically measures cell viability by detecting the formazan product, which is formed when mitochondrial enzymes in living cells reduce MTT. The standard wavelength for reading the absorbance of the formazan product is around 570 nm (sometimes 560-590 nm depending on the protocol and instrument).
If you read the plate at 540 nm instead of the standard 570 nm, the absorbance readings may not accurately reflect the amount of formazan produced, potentially leading to inaccurate measurements of cell viability. The absorbance at 540 nm is likely lower than it would be at 570 nm, which could underestimate cell viability.
To ensure credible results, it's advisable to repeat the assay using the correct wavelength. If that's not possible, you could potentially calibrate or normalize your results by comparing them with a standard curve generated at 570 nm, assuming you have access to one. However, this would still introduce some level of uncertainty.
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