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Cell lines behaving differently to drugs

MTS assay

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2 replies to this topic

#1 Peniel

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Posted 14 April 2015 - 01:50 AM

Hi eveyone,

 

I am in such a dilemma. I did a dose-response curve for some inhibitors in some cell lines and generated IC50s of the inhibitors.

 

I calculated the IC25 from the IC50 in other to combine with the IC50 of another inhibitor previously generated. The IC25s seem to inhibit about 50% of the cells now and the IC50 inhibits about 60-70%, Is there an explanations for this. Has anyone come across such a thing.... It is driving me nuts as I don't know how to explain the results.

 

Thanks for your help.

 



#2 DRT

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Posted 14 April 2015 - 12:07 PM

I don’t think you need to be too worried about variations of this magnitude as long as they are random ie not continually increasing, and the difference between the two inhibitors is roughly the same. Subtle changes in the cell line or the serum used in the media could alter the inhibitor response in ways that you would not see if you had text-book pure receptor and inhibitor.

 

Having a greater error associated with either the IC50 or the slope of the dose response curve will affect your ability to determine a ‘significant’ interaction between the inhibitors but you will be able to separate out the intra- and inter-assay variations. {Sorry that doesn’t sound very clear.} I guess I’m recommending that you do a full dose response for both inhibitors and any combinations each time you come to repeat the assay. That way any variation in the slope of the dose response can be incorporated into your calculations.



#3 Peniel

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Posted 27 April 2015 - 02:14 AM

Thanks for your help DRT. It is difficult to do a full dose response every time using the MTS assay because the reagent is really expensive. I am attributing the variation to the different lot numbers of the inhibitor which could mean it is more potent or like you mentioned the cell lines might have have changes from being passaged.

 

Can I ask another question please? When doing cell signalling experiments which is the best method to harvest......trypsin or scraping? and why? I am worried about cell surface receptors. 

 

Thanks a lot!!!







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