MTT assay with polysaccharides - different results every time. Help! - (Jul/16/2013 )
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!
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.
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.