Why not separate the assays with 2 readouts instead of one? Or try a Cell Cycle assay using flow cytometry.

Hi Scott, thanks for the reply and I cannot agree more. I have run flowcytometry but only 7aad single staining and the results was so puzzling which was why I redo the MTS. Previously I used nasopharyngeal carcinoma, NPC cell line from my own lab and there was only a single point which seems like an outlier and I processed the MTS data using GraphPad which might not take that single data point in account. The Plk1 inhibitor IC50 was at microMolar which my supervisor and I was quite surprised because we expected it to be at nanoMolar range as other papers have shown. But we went on with the data for a while (which was a grave mistake) until I managed to do an attachment in a research institute. Using the same cell line, the data showed 2 points that caused the U-shaped and I cannot just assume that they are outliers. So now, instead of just blindly using graphpad, I am trying to find if there is any way to get the correct IC50. Until just today, matter of fact, couple of hours ago I found a paper "Mitotic arrest and slippage induced by pharmacological inhibition of Polo-like kinase 1", it really explains everything. Somehow at high concentration of Plk1 inhibitor, BI2536 caused mitotic slippage. I have also used RO3280, another Plk1 inhibitor as a control and the same pattern surfaced. I am planning to run PI staining soon to prove that was what happened (high number of 4n population). So far there has been no paper that really tried high dose (they just try 100 - 10nM range) which was why I did not find possible explanation, prior to PI staining. With the 7AAD staining, not enough information can be obtained from that and caused me a real headache analyzing the data. Based on that paper, my concentration used for 7AAD was at micromolar range which is just general toxicity towards normal immortalized NP and NPC cell lines. I was expecting to see low cell death in normal but high in NPC but I got both high cell death. Based on the new MTS assay on normal NP, it is 3-fold less sensitive (using points under 100nM to calculate the IC50) compared to NPC. Anyway, thanks again for the suggestion.

Hi Reagen,

Can't offer too much help about the assays, but biphasic will not work for your data (biphasic needs the drugs at similar effects at both curves, e.g. 100% to 50%, then 50% to 10%), as your drugs appear to have opposing functions at various concentrations (e.g. drug A at high nM conc appears to offset its own effect at low nM conc). A possible better model will be bell curve (http://www.graphpad.com/guides/prism/7/curve-fitting/index.htm?reg_bellshaped_dose_response.htm), which will give you EC50 1 and 2.

Hi Jacky, thanks for the suggestion. I have tried using biphasic and u shaped model of graphpad before but the program will only be able to fit in the graph if the last point at the high concentration is taken off. The program could only fit a bell shaped curve from the peak of the lowest concentration to the peak of the highest concentration. 

I would thoroughly work with a colleague's opinion especially with colleague Reagan Entigu who gave a very clear and documented acceptance to measure pharmacokinetics - an analysis of biphasic property (IC50 for drugs that have biphasic property)! Very useful for me!

Thanks Jasenko. As I am using biphasic drug, i have managed to find a combination with another targeted drug to lower the concentration of BI2536 under 10nM. But the question remain, should a biphasic drug be suitable or better yet 'a good candidate' for future treatment? There are papers saying that it should be use cautiously as it may accelerate cancer growth. Most of the papers that used BI2536 used under 100nM, not many has tested above that. It is just for my own knowledge should I be asked in viva. Though my study would be focusing on to better understand the underlying survival and apoptotic mechanisms that are shared by NPC cell lines due to heterogeneity rather than to provide a final cure for NPC which will take much in depth study.

You welcome!Thanks also for this additional explanation that will be very useful for my education! That is why I have recommended all your answers with great pleasure!

Regards from Winter Sarajevo 

Jasenko