In a recent article published in Nature’s Laboratoy Investigation journal, we demonstrated that drugs with anti-oxidant properties can interfere with cell viability measurements by assays that rely on the reducing property of viable cells, giving spurious results. The current manufacture recommended protocols of the assays assume that the assay reagent is almost entirely reduced intracellularly in viable cells. The potential for direct reduction of the reagent substrate by the drug being tested is somewhat underestimated (with the test compound control stated to be 'optional' in the Cell Titer Blue protocol). Given that these assays are convenient and widely used, this can have significant implications with indigenous compounds/ drugs having anti-oxidant properties and being screened for their cytotoxic potential against cancer, running the risk of being discarded due to spurious results. The article also demonstrates that by incorporating simple modifications in the protocol, these spurious results can be avoided.
Here-in, the modified Cell Titer Blue assay protocol is discussed step-by-step. The specific modifications added to the manufacture recommended protocol are in bold font. The rest is as stated in the manufacture recommended protocol.
The modifications can also be incorporated in other cell viability assays that rely on the reducing property of viable cells such as Alamar Blue assay, MTT assay etc.