I did PrestoBlue cell growth and proliferation assay and the numbers of drug group is smaller than the medium so the treated group-medium is negative. Does this mean all cells died ? But under microscope, there are still cells.
Dear Heba,
I didn't use presto blue before but have used EZBlue extensively for cell proliferation. Please clear my doubt. By medium you mean culture media alone?
If that's the case then your read should be always more in cells treated with group..
But if it's cells with culture media and cells with treatment, then the read can be lower as drug can also induce cell death and hence a lower read as compared to control group.
But as you are saying negative reading, that can't be there.....You have to give longer time incubations as some cells take time to convert the dye..If your treatment reading is lower than the media alone without cells, there is some technical error. Practically it's not possible and it shouldn't happen..
Hope it helps !
Yes ajay. Medium without cells for background noise, it is higher than the treated group and this is error.
Dear Heba,
probably it's a background problem. Here's some hints from PrestoBlue FAQs.
Regards,
Fabio
What could have caused high background fluorescence values in my PrestoBlue
® assay?
Background fluorescence can be corrected for by including ‘no cell’ control wells on your assay plate and subtracting the average of those fluorescence values from your assay wells. There are a few factors that may lead to high background fluorescent values.
1. Prolonged Exposure to Light: Extended light exposure will break down resazurin, the active ingredient in PrestoBlue® reagent. Always store PrestoBlue® cell viability reagent in the dark and do not expose the reagent to direct light for long periods of time (see Figure 5).
2. Contamination: Bacteria will reduce resazurin to resorufin. If the reagent is compromised, it should be thrown out and replaced.
I have extensively worked with MTT, WST-1, and XTT reagents for cell growth assay. It is not normal to have the absorbance of your medium alone higher than that of treated- even if your treatment kills cells. Are you sure your medium is not contaminated? And for how long did you incubate the dye? What were the absorbance values of Control group? If the problem persists, I suggest you switch to another cell growth assay, such as MTT or WST-1...
Hope this helps,
Patrick
- Badges
- Science method