Cell proliferation/viability assay - use of WST and 1-Methoxy PMS (Oct/15/2005 )
I'm trying to set up cell viability assay for the lab. Upon searching around, I found using WST seems to have more advantages over using either MTT or XTT. However, there seems to be a whole variety of WST available in the market (e.g. WST-1,WST-3,WST-4,WST-5,WST-8,WST-9,WST-10,WST-11,WST-20,WST-21,WST-22). I'm wondering as which WST I should go for, in terms efficiency and accuracy. Thanks for any of your suggestions.
Additionally, with the MTT assay currently used in our lab, the protocol claims to use a single wavelength (492nm) for the detection. However, I've also found other protocols using 570nm for the detection and 670nm for the subtraction of background. Could anyone tell me are these two protocols all valid? And, what if we don't subtract out the background with the 670nm readings? Will this seriously affect the accuracy of the MTT assay at all? Thanks.
I have no experience using WST, although I can recommend MTS from Promega if you're interested in a soluble compound that is easy to use.
As for your question on background measurements, I think subtracting background would increase the accuracy of your measurements, although the end result may not be much affected if the samples are normalized to a negative control. Follow the protocol supplied with the compound you decide to purchase. Subtracting the 670nm measurement would be fine, so long as the compound has no/minimal absorbance at that wavelegnth in either the original or reduced form.
Our lab has used MTS quite a lot to screen compounds for activity. The background subtraction at 670 doesn't hurt, but should not be necessary if your plates are clean (ie: give them a wipe before putting them in the reader).
We have found that the MTS solution "goes off" sometimes, leading to results of an almost random number quality. We have therefore switched over entirely to the Cell-Titer blue indicator (also from Promega). Not only is it cheaper, but it works a lot better - less background noise and more reproducible results. If you have a fluorescence plate reader, I highly reccomend it.
We use WST-1 every so often in our lab. It's a nice reagent because it is simple to use ie...add to cells and wait 4 hours before reading.
As it measures aerobic metabolism however, that may not necessarily correlate with viability. My colleague tried to use it to measure T lymphocyte viabilty and it appeared that they were dying with the compound treatment, but really it turns out that they weren't actually dying by other methods. Apparently, activated T lymphocytes just switch to glycolytic metabolism.
We tried the Cell-titre glo from Promega to test the viability and it works just fine. Grow cells in white optical plates for required duartion and add the reagent and read the lumisence in 10 mins. What sucks is it is expensive.