wst1 blank absorbance problem - (Apr/03/2014 )

Hi everyone,

Recently, I've tried wst1 assay for the first time. After treating the cells with chemical, i added 100 ul fresh DMEM containing 10 ul wst1 and incubated for 3 hours. I've measured the absorbance in 355-460 interval (this was the closest filter pair to 450 nm -that I could choose in our reader-). Unexpectedly, I got the maximum absorbance values in my blank samples (DMEM+wst1; without cells) and positive controls (detergent treated cells with DMEM+wst1;suppose to be all dead!).

In parallel, the lowest absorbance values were in my negative control samples (untreated cells).

I really couldn't make any sens of this! Has anyone faced such a problem and do you have any suggestion?

Thank you!

I suspect that you will probably need filters pretty close to the absorbance recommended by the protocol.  This is essentially the same as an MTT/MTS assay as far as I can see, so there are heaps of threads on here about those - have a look, someone else may have struck your problem too.