I am currently working on MTT assays for determining the molecule IC50 on cells. Based on the MTT results, I want to determine IC50 in GraphPad using the raw data "concentration (not log[concentration])-%cell viability". For this, is it appropriate to calculate IC50 by normalizing the min and max Y value as 0-100 without transforming my concentration data to log derivative and selecting the Nonlinear regression->Dose-response-Inhibition-> "[Inhibitor] vs. normalized response" model from XY analysis?
According to the protocol written on GraphPad's website, it transforms the concentration as X=logX, normalizes the min and max Y values as 0-100 and when I select the Nonlinear regression->Dose-response-Inhibition-> "log(inhibitor) vs. response--variable slope (four parameters)" model from XY analysis, it gives the warning "very wide" for the IC50 value. I get the same problem when I follow the above steps using "Concentration-Absorbance" raw data.
Could you also explain which of the equations in the "dose-response-inhibition" section in the picture attachment would be more appropriate for my study? And, which steps should I follow to calculate the IC50 value in GraphPad?
Thank you for your concern and answers, wish you all happy new year :)
If control mortality is >10% then you do need to correct the data using the Schneider-Orelli variant of Abbott's formula. With dose responses it is usual to have the log of the concentration although you could use a non-linear model. With Graphpad I tend to avoid feeding it raw data as it is quite a fussy program.
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