I know the Alamar Blue assay measure the redox reaction of metabolically active cells and when reading the plate a higher fluorescent value is supposed to correlate with higher metabolically active cells. In trying to test if a protein creates a concentration dependent or time dependent toxic effect to cells, we found in control wells with no cells and our protein the plate reader can give off values similar to a cell viability of 100%. Why is this?
Hello Danielle Moss
Please refer to the research article attached below.
Article Alamar blue reagent interacts with cell-culture media giving...
It states that the reagent can itself interact with the media causing significantly different fluorescence over time in a manner independent from the effect of intracellular reducing environment of living cells on alamar blue.
In addition, their results indicate that fluorescence varies as a function of incubation time with the reagent. These findings signify the need for routine subtraction of the background fluorescence of media-only with alamar blue reagent during measurement of cell viability by this method in order to determine an accurate measurement of cell viability.
Other possibilities could be:
1. Alamar Blue reagent being exposed to direct light for extended time.
2. Check the instrument settings.
3. Longer incubation time.
4. Bacterial contamination in the culture media.
Best.
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