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How to know best concentration for motility assay


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#1 dnasequencer

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Posted 26 April 2017 - 03:31 AM

Hi,

I am working on a curcumin derivative and found that 5uM is cytotoxic and inhibits MDA cells proliferation.

The next step is to do scratch assay, but I am wondering can I use the same dose 5uM, although this might kill the cells and prevent their motility.

Any suggestions?

 



#2 bob1

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Posted 26 April 2017 - 06:39 AM

I would say no - cytotoxicity and prevention of proliferation are two different things - if could be that the loss of proliferation you are seeing is actually death of the cells not an inhibition of growth. You need to do an EC50 test to determine the effective concentration. 



#3 dnasequencer

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Posted 27 April 2017 - 12:34 AM

Thanks bob1

I did proliferation assay and cytotoxicity assay. The effective concentration starts from 5uM, so obviously I will get different EC50 for each assay.

Which EC50 I should use? and is this EC the one I can use for the motility assay.



#4 bob1

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Posted 27 April 2017 - 06:10 AM

If the effective concentration is 5 uM and it is cytotoxic at that level, then it is highly unlikely you can use it for anything drug wise - it will kill the cells. This is fine if you want to kill cells (including "normal" cells), but it isn't if you want to do anything else. The Ec50 should not vary based on assay - it should give you the same value (+/- a bit, there is variability but it shouldn't be a log10 out). To do an effective EC50 you should have 10-15 data points from difference concentrations of drug, some above your concentration and some below, then have plotted the sigmoidal curve. If you haven't done this, then moving on to other assays is a bit pointless.

 

Like I said above, inhibition of proliferation and cell death are two different things - if you can't measure loss of proliferation without encountering cell death then there are no assays that will work.



#5 dnasequencer

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Posted 29 April 2017 - 11:43 PM

Thanks a lot bob.

It is clear now.






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