The Use of Simultaneous Fluorescence and Differential Phase Confocal Microscopy to Study Alamar Blue™ Reduction in an Epithelial Cell Line

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Abstract
The Alamar Blue™ reduction assay is used as an indicator of cellular viability in in vitro tests for the prediction of ocular irritancy. Alamar Blue itself has a low cytotoxicity, so repeat exposure and recovery studies can be performed on the same cells. This paper contains the results of a preliminary investigation of interactions between the Alamar Blue dye and a confluent monolayer of epithelial Madin-Darby canine kidney cells. This was performed by using an experimental fluorescence microscope and differential phase confocal microscope designed for studying live samples in vitro. The initiation of Alamar Blue reduction to its fluorescent product did not occur at the same time in all cells, but started in a small number and spread progressively through their immediate neighbours. There was clear localisation of the reduced (fluorescent) Alamar Blue within the nuclei and other organelles.