Hello everyone!
I am testing the effect of certain compounds on cell proliferation by MTT assays. We usually analyse the data using Excel followed by Graphpad prism in our lab. I sometimes get complete, near-to-perfection sigmoidal curves & sometimes it is just incomplete half curves which are not sigmoidal and end somewhere halfway down the Y-axis. Does anyone know why does this happen? Any suggestions to get proper sigmoidal graphs? Thanks again!
It's hard to say without seeing your curves, but you most likely need more concentrations and more replicates (less error) would probably be good too. The more concentrations you use the more perfect your curve should become. The lowest concentrations should barely affect proliferation and the highest concentrations should completely stop proliferation and/or kill all cells. There are also options in Prism to constrain the bottom and top of the curve (i.e. to 0% and 100%) which you could try, but be careful because that affects your calculated IC values.
Like Nathaniel said, without an example curve it's hard to tell what the problem is. However, have you checked whether your numbers migrated from Excel to GraphPad are still recognized as numbers in GraphPad? Having had multiple issues in the past with the German system (using semicolons instead of full stops and vice versa for number display) I have sometimes encountered similar graphing issues.
In addition, you might want to check whether your axis scale and data are compatible to each other.
I haven't seen your curves, but an approach might be to try ten-fold dilutions with your compound (from very high to very low doses) so that you can have an idea about the effects in a broader concentration range and also might get a sigmoidal curve.
Dear Shruthi,
Have a look to this simple guideline. It may help you
Furthermore, It is not easy to get every time the perfect curve in the MTT assay. It requires high precision to do that. As the respectful researchers said above, It is better to provide us with example of your work (curve and results)
Regards
Hello everyone,
Thanks for responding. I am so delighted that you've given me your valuable time in trying to answer my query.
Well, I've done many MTT assays with Temozolomide and Cisplatin in the past (my Mentor's suggestion to work with established compounds to develop a technique for doing the MTT assays). I have now moved on to new compounds. for which I have to determine the IC50 values in different cell lines.
I am attaching a word file with the dose-response curves I've obtained using a test compound and here are the various test parameters:
Cell plating number: 1000 cells/well
concentration of the stock compound: 100mM
Post-drug addition incubation time: 72 hours
These are the dilutions I can work on (cannot increase the # of dilutions more than this)
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