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 sense of this. Has anyone faced such a problem and do you have any suggestions?
Hi,
check for microbial contamination as it can produce readings you described.
Hi, I repeated the experiment and still got the similar results. This time I'm sure that there is no microbial contamination. I still have the similar pattern; lowest absorbance in the control cells-which are alive, I also checked with microscope they look pretty healthy-, and the highest in the death cells. Even more strangely, I also measured the untreated cells -no wst1 added- in the same plate with my samples and they gave even higher absorbance (about 6 fold of the control).
I'm really confused, can anyone help?
Hi, have you solved this problem?Because I have the same situation and I don't find an explanation. Thank you in advance.
Hi Francesca,
I did, actually it way a silly mistake. I've realized that I was using the wrong reader -fluorometric - for measurement. The problem was solved when I used the colorimetric plate reader.
Good luck
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