THE USE OF SIMULTANEOUS FLUORESCENCE AND DIFFERENTIAL PHASE CONFOCAL MICROSCOPY TO STUDY ALAMAR BLUE(TM) REDUCTION IN AN EPITHELIAL-CELL LINE

The Alamar Blue(TM) reduction assay is used as an indicator of cellula r viability in in vitro tests for the prediction of ocular irritancy. Alamar Blue itself has a low cytotoxicity, so repeat exposure and reco very studies can be performed on the same cells. This paper contains t he results of a preliminary investigation of interactions between the Alamar Blue dye and a confluent monolayer of epithelial Madin-Darby ca nine kidney cells. This was performed by using an experimental fluores cence microscope and differential phase confocal microscope designed f or studying live samples in vitro. The initiation of Alamar Blue reduc tion 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 reduce d (fluorescent) Alamar Blue within the nuclei and other organelles.