Hi Bora, I agree with Vraj with the exception that it is far more robust to estimate ED50 after non-linear regression/fitting of a sigmoid to all the experimental results instead of using only the "linear" part of it (and you can use Excel to do it, no need to specific software).

Additionally, you need to choose properly the chemical/drug concentrations, otherwise you may end up failing important portions of the dose-response curve (too low or too high conc's). It is also common and advisable to use a negative control.

Hope it helped.

BR Luis

BTW, a general RG community rule is to upvote any contribution you find helpful (also to downvote the ones you find being wrong or misleading).

To get ED50 (effective dose to create 50% change in specific parameter) of DMF in your cell line, incubate various DMF concentrations (e.g. 0, 5, 10, 20, 40, 80, 160 µM) in different test reactions (e.g. different wells in a 96-well plate) containing equal number of cells (e.g. 5 million/well) for a defined time (e.g. 4 to 24 h or depending the sensitivity of your cells to DMF). After the incubation, read your specific end-point (e.g. cell #, cell viability, or any relevant marker of your experiment). Finally, plot a graph log concentrations (on X-axis) vs your % read-out (e.g. %cell #s relative to control, i.e. cell numbers observed at 0 DMF concentration in the same experiment). Now draw a regression line through a linear portion of your graph (usually 20% to 80% of your control). The ED50 of DMF for your cell-line can now be easily calculated from the slop and intercept values of your linear regression analysis.

The endpoint calculation can be done by non-linear methods or software assisted algorithms but make sure that you are using data from the linear phase of your regression curve. Hope this helps!

Hi Bora, I agree with Vraj with the exception that it is far more robust to estimate ED50 after non-linear regression/fitting of a sigmoid to all the experimental results instead of using only the "linear" part of it (and you can use Excel to do it, no need to specific software).

Additionally, you need to choose properly the chemical/drug concentrations, otherwise you may end up failing important portions of the dose-response curve (too low or too high conc's). It is also common and advisable to use a negative control.

Hope it helped.

BR Luis

BTW, a general RG community rule is to upvote any contribution you find helpful (also to downvote the ones you find being wrong or misleading).

Thank you so much for your  helpful answers! I'll try to implement what you said on my calculations Thanks again 

Hi Bora,

I concur with Luis on this. Yes, non-liner fitting are much more robust. I probably oversimplified my earlier answer! Thanks, Luis for remanding that we can also calculate EC50s by Excel. Usually we use STATISTICA for fitting sigmoidal or other non-liner equations to all the experimental results.

Also, I agree with Luis on the NCE concentration test range. If you do not know the target range of your class of NCEs, common approach is to do 10x dilution (e.g. 0.01, 0.1, 1.0, 10, 100 uM) in a preliminary experiment and then based on that data, in final experiment you can set refined range of concentrations to see a nice looking graph!