I have cancer cells overexpressing GFP and compared their proliferation and viability to the wild-type cells. MTT assay shows an increased signal for the cells overexpressing GFP. However, other proliferation assays did not show this effect. I repeated the experiments at least 3 times and the results have always be the same. How can this be happening? Are there potential metabolic change going on? Or do the cells just don't proliferate so fast but are still alive?
Can you give more information on the other proliferation assays you used?
You can probably find the answer to your question if you read the principle of these different assays.
For example, the MTT assay measures cell viability by assessing the mitochondrial ability to metabolize MTT into Formazan. It therefore largely relies on the cellular (mitochondria) metabolic activity of living cells. You can sometimes get different results if you compare MTT assays with other assays which use different approach to measure cell proliferation (e.g., PI assay measures cell proliferation by binding to DNA).
So, your results may be impacted by the main cellular target (cytoplasm? mitochondria? nucleus?) of your experimental compound.
There might be a role for GFP on cell activity, and potentially interfere with MTT.
Check out this publication:
Article GFP Affects Human T Cell Activation and Cytokine Production ...
Marius Baguma : The other proliferation asssay was just simply seeding a certain amount of cells into 96 well plate and monitor the prolifewration for 5 days. The growth curve and the doubling time of the cells was than calculated from these data.
Dear Vanessa Krauspe , how did you quantify the proliferation? Just by observing your cells on a microscope ? Have you taken some measures during this monitoring? I guess yes, otherwise you would not be able to draw a growth curve.
Dear Marius Baguma, proliferation was monitored by the IncuCyte and the software than calculated the growth curve according to the data.
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