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MTT assay with polysaccharides - different results every time. Help!

MTT assay Wst assay

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#1 Elioelio

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Posted 16 July 2013 - 11:49 AM

I'm a PhD student and have been using Wst and MTT assay for a couple of years now. I am working with pectins - some are purified, while others are pretty crude.

Take one of the purified pectins for example: I have carried out many Wst and MTT assays. I'd say that ~80% of the time the pectin reduces cell proliferation ~(according to the assays). Because this doesn't happen 100% of the time I'm confused! And the amount it reduces proliferation by varies also - it can be between 10-60% reduction.

This happens with the other pectins too - although the times they reduce cell proliferation could be 50/50%.

I use FTS (cancer drug) as a control and get similar results every time.

I could say that the cruder pectins may not be homogenous and so act on the cells differently every time. Could I say this about the purified pectin too?

Its driving me crazy! I so want to move on, but every time I do an assay and the pectin does not reduce cell proliferation, I feel I have to do it again, and again... and again.

I realise most scientists would just do the assay 3 times and move on... But I can't seem to.

Anyone with experience working with polysaccharides and cell proliferation assays?
Or any info or ideas are appreciated!

#2 DRT

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Posted 16 July 2013 - 01:12 PM

Are you being particular about the condition of the media as you add the pectin? I can imagine that small changes in the pH make a large difference to the solubility/gelling/interacting properties of pectins.

Also, have you tried generating a full inhibition curve ie reporting the IC50 ±SD rather than trying to hit a single concentration? If the inhibition curve is steep then small variations in IC50 will cause a large difference in the %inhibition at a single concentration.

#3 Elioelio

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Posted 17 July 2013 - 01:25 AM

I've done 3 concentrations. Almost always it is dose dependent. I've not worked out the IC50, but thanks, thats not something I've thought about before.

With regards to the media - I add all the pectins to the same media. Some react differently - the low de-esterified pectins become more gel like as they react with the calcium. But the rest - there seems to be no difference. All the pH's of the pectins are different, but all go to pH8 in the media.





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