Bleaching out of the pink color of the reduced state of the dye (quenching of fluorescence) usually occurs after very prolonged incubation times due to the formation of a colorless product called dihydroresorufin. Exhaustion of the buffering capacity of the dye’s formulation may explain the formation of this product. 10% fetal bovine serum (FBS) and bovine serum albumin (BSA) may also cause quenching of the assay [9,34]. Phenol red shifts all values 0.03 units higher than without phenol red, but it does not otherwise affect assay chemistry. Microbial contamination will reduce the dye and lead to false positive signals hence, the assay must be carried out under aseptic conditions and the medium may require antibiotics to eliminate microbial contamination