Not sure what caused that kind of result. The MTT assay was conducted using instructions and then the absorbance of final formazan product was analyzed at a certain wavelength (570 nm) to get the raw data absorbance values.
Then a graph was made up in the Prism based on the percentages that were calculated from the raw data absorbance values. A Log Scale was then used to calculate IC50, so the graph was transformed and then analysed one more time ( ‘Dose Response – Inhibition’ and then ‘Log(inhibitor vs. response – variable slope (four parameters).’).
How can I understand why is there >100% of Cell Viability and also
Try the MTT assay measuring the absorbance at 490 nm (Berridge and Tan 1993).
Aleksandra Ivanova, the cellular viabilities above 100% are the result of an erroneous data that was included in the calculation of the average of the control. The raw data of the control are: 1,978, 0.617 and 2.378. The value 0.617 is very low, and this induces an average for the control lower than it corresponds. In this way, some percentages of viability obtained at non-inhibitory concentrations of the toxicant result falsely higher than the control. My recommendation is to discard the wrong value (0.617) and recalculate the average of the control with the remaining data.
I would not worry about negative results very close to zero. The interpretation that must be made is that [toxicant] > 10 units destroy 100% of the cells. However, one possible explanation is that the blank wells have been prepared with fresh culture medium (did you do so?). In this case, it should be remembered that during the incubation of the cells treated in humid atmosphere of CO2 at 37 °C, the pH of the culture medium becomes acidic and the color of the medium tends to pale and absorbs less radiation; on the contrary, a fresh medium can absorb a little more. This difference in color absorption could be avoided if the blanks are incubated under the same conditions as the cells before the MTT assys.
On the other hand, I note that your graph does not include the results of the control and the two lowest toxicant concentrations. About this I attach an Excel file where I do all the calculations with your data and I make some suggestions to graph and be able to include all the results.
Finally, in some cases your data, within the same treatment, have an important dispersion (see error bars on graph of attached file), I recommend to check the technique of pipetting and homogenization of cell suspensions.
Patrick Heinrich gave a clear explanation about viability over 100 percent in MTT assay. I highly recommend his answer to an old question in researchgate.
You could find it here: https://www.researchgate.net/post/What_are_the_reasons_for_cell_viability_of_more_than_100_for_DMSO_control_vehicle_in_MTT_assay
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