I have a problem in which after finishing the MTT assay and start to take the results , the absorbance comes from the plates is not trending in which the highest concentration gives availability higher than the lower concentrations of the drugs I have used ?
Hello,
As what I have understood from your question is that highest drug concentration gives higher absorbance in MTT assay. If this is so, then there are two possibilities.
1. The candidate drug may be causing an increase in the activity of the enzymes (mitochondrial enzymes) without causing an increase in cell number.
2. The candidate drug may be causing cell proliferation.
In both the above cases you could get high absorbance reading In MTT assay at high concentration of the candidate drug because of increased formation of formazan crystals.
In addition to MTT, you should also carry out Trypan Blue exclusion assay to check percent viability with similar drug concentrations. This might help.
Best,
I agree with Malcolm Nobre . I had a problem that MTT results showed almost no reduction in cell count after treatment but I could see looking significant cell death under a microscope. I did Trypan blue staining which showed significant decrease in cell count. The difference in result was 80% live cells in MTT and 25% live cells in Trypan blue method. This discrepancy happened because my treatment caused increased mitochondrial activity which lead to more formazan formation in MTT assay.
@Malcolm Nobre , @Mateo Glumac Thank you very much , I will try to carry out Trypan Blue exclusion assay.
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