Cell-based assays using calcein acetoxymethyl ester show variation in fluorescence with treatment conditions
Fayth L Miles1, Jill E Lynch2, Robert A Sikes2
1Department of Epidemiology, Fielding School of Public Health, University of California, Los Angeles, Los Angeles, CA, USA; Laboratory for Cancer Ontogeny and Therapeutics, Department of Biological Sciences, The University of Delaware, Newark DE 19716, USA; The Center for Translational Cancer Research, The University of Delaware, Newark, DE 19716, USA.
Abstract:
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.
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