I have used the MTT Assay to measure cancer cells' viability under an antioxidant compound's influence. But contrary to expectations, with the increase of antioxidant concentration from 5 μM to 150 μM, the viability not only did not decrease but also increased. In other words, with the increase in the concentration, the amount of light absorption increased. In your opinion, what is the reason for this technical error? Or what kind of problems could have occurred during the MTT Assay?
if you are looking at viability, did you use any other methods? mtt depends on mitochondria. and you are using antioxidants.
This is completely normal, antioxidants promote the viability and proliferation of tumor cells. (please see also the Warburg Effect).
Yours,
Thanks to both professors Luke Parkitny and Mourad Aribi for answering this question.
In fact, we want to measure the expression of several microRNAs and LncRNAs in a cancer cell line under the influence of an antioxidant, and for this we need to choose one or two concentrations of antioxidants and measure the expression of genes accordingly. Therefore, We used MTT Essay.
In your opinion, What other methods are there to reach the final concentrations?
Dear Dr. Mourad Aribi
I appreciate your valuable comment, theoretically, you are right, but don't you think the antioxidant itself interferes with the MTT dye?
Hi Navid. As antioxidants treatment would reduce the effect of oxidant species, which some of them trigger signaling pathways that lead to cell death. Therefore, it is acceptable that antioxdants increase cell viability.
However, if you're still suspecious about your expecations, make sure your treatment does not have any significant absorbance at 500 - 600 nm, and when you see your cells under microscope, you find those with highest concentration of treatment, the happiest, and vice versa.
Hi dear Dr. Mohammad Pirouzfar
According to related articles that have been done recently in the field of this study, in the range of 5 to 200 μm using the MTT test, they have reached IC50=80 μm, which means that with the increase in antioxidant concentration, the amount of viability has decreased. But my Mtts in 24 and 48 hours are the opposite of the results of these articles, that is, the survival rate of cancer cells increased with the increase in concentration. Therefore, I suspected that the MTT dye interfered with the antioxidant itself, and when I added the MTT dye to the antioxidant concentrations in the absence of cells, I found exactly that the MTT dye interfered very strongly with the antioxidant, so I decided to use the PI staining method instead of the MTT method. Of course, before that, I want to observe the effect of antioxidant effect on cells with the trypan blue method.
Thank you for your valuable and useful comment
Yours sincerely
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