Cell-based assays using calcein acetoxymethyl ester show variation in fluorescence with treatment conditions.

The use of fluorogenic compounds in cell and molecular biology has increased in both frequency and range of applications. However, such compounds may introduce artifacts in intracellular fluorescence and cell number estimations as a consequence of interaction with exogenous stimulants, necessitating the use of adequate controls for accurate measurements and valid conclusions. Using calcein acetoxymethyl ester (AM) in combination with various exogenous cellular treatments, we report that the standard practice of direct normalization of experimental values to controls is insufficient for fluorogenic measurements. Treatments applied to cells may influence intracellular conversion of the fluorogenic compound, thereby enhancing or decreasing fluorescence relative to controls. We hereby encourage caution and recommend normalization of cellular fluorescence within each treatment group before comparison to controls.