Hi, guys. I've been doing MTT experiments for a few years. And i've always had one question about the background reading of the MTT, usually at 630nm. You can find a lot of papers in which the authors had done it, but you can also find a lot of papers without this kind of measurement.
What would be the importance of that background absorbance reading? Does your lab do that kind of reading? How you guys do it? Do you subtract the background value from the absorbance of the experiment? Never found those answers in scientific papers.
Thank you!
Dear Julio Thurler
The background absorbance reading at 630nm is usually used to correct for optical variation. It allows individual corrections to be made for each experimental well, due to for instance, a scratch or any accidental fingerprints that may arise on a 96-well plate by holding the plate on the long sides between thumb and fingers. This would change the background absorbance value of that well relative to the unmarred wells next to it.
As you mentioned, a lot of papers do exist without this kind of measurement. It won’t make much of a difference in the final reading if you wish to carry out MTT assay without carrying out the background reading at 630nm provided you have enough replicates in your experiment and handle the plate properly while performing the MTT assay as well as the wells in the 96-well plate should not vary in quality (a non-defective 96-well plate).
Nevertheless, if you still wish to take the absorbance at 630nm, then you should subtract the 630 nm background absorbance from the 570 nm measurement.
Best.
Dear Julio,
You can add that you need to subtract the measurements at the main wavelength of 570 nm measurements at the reference wavelength of 620 nm.
Best regards,
Alexandr
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