The optimum pH of the assay is ranges between 7.0 and 7.4, therefore, culture medium must have
buffering capacity. The optimum incubation temperature is 37 °C and plates must therefore, be sealed to prevent evaporation. Assays must be carried out at a uniform temperature to ensure reproducibility of all wells and across a single plate. Alamar Blue is photosensitive and the incubations must be done in the dark. The culture medium and the test compound themselves should not interact with the assay chemistry. Negative and positive controls must be empirically determined to ensure that there are no non-specific interactions with assay chemistry which would result in artifacts or false positive signals. The incubation time and cell density must be empirically determined and standardized as low cell density means slower growth and lower than expected levels of dye reduction. The end-point of the assay depends on the cell density used. Generally, it is recommended that the cells should be in the exponential stage of growth. It is also preferred that the culture medium should be synthetic and defined, however, it must also allow sufficient growth so that stimulatory or inhibitory effects of test compounds are not exaggerated or underestimated