Elflick

I am a PhD student testing the bioactive effect of various polysaccharides on colon cancer cells. I am using the wst-1 cell proliferation colorimetric assay to assess whether the pectins are killing my cells. I add the pectins in medium onto adhered cells, incubate for 48hrs, then add 10ul of wst-1. WST-1 is a tetrazolium salt that when in contact with metabolically active cells gets cleaved to formazan by mitochondrial dehydrogenases. I then mix the plate for 5-10mins and read on a plate reader. I have been taking readings every 2mins from 10mins to 1hr, then every hour after that My main question is this: How do you know when the best time is to read the absorbance?? Is 10 minutes long enough for the reagent to work? The instructions say that the time depends on the set-up, but to test at 0.5 – 4hrs. The problem is that the reagent seems very sensitive as, for example, at 10mins a pcompound will be significantly different from the control, but at 15mins will not be. Or will be sig diff at 30mins but not 10mins. Then another compound on the same plate will be sig diff at a totally different time point. My supervisor is saying that if it is sig diff at i.e 10 mins then this is proof that the polysaccharides are killing my cells, even if it is not sig diff at 15mins. I have been taking readings at several different time points, and taking the most significant readings for each of the compounds, and normalising the absorbance reading to the control. I have been getting fairly replicable results. However, I am not totally convinced. Surely the longer the Wst is left, the more accurate, as the dehydrogenases will have had time to cleave the Wst. There will be a time point where the Wst will be saturated, but I tested at 4hrs and the absorbance was still going up. There is the added issue of the larger polysacchrides aggregating and not allowing so much of the wst reagent though? I am mixing for 5-10mins so I am hoping this is not an issue... If anyone has any thoughts on this please let me know! I have been doing this assay on and off for 8 months now and I'm worried that my results may not be totally valid!