ATLA::Alternatives to Laboratory Animals

Volume 26, Number 4

The use of simultaneous fluorescence and differential phase confocal microscopy to study Alamar BLue™ reduction in an epithelial cell line.

ATLA 26, 505-522, July/August 1998

John R. Garside,1 Richard H. Clothier,2 Mike G. Somekh1 and Chung Wah See1

1 Department of Electrical and Electronic Engineering, University of Nottingham, University Park, Nottingham NG7 2RD, UK; 2FRAME Alternatives Laboratory, School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK

SUMMARY

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 localization of the reduced (fluorescent) Alamar Blue within the nuclei and other organelles.

Keywords: Alamar Blue™, in vitro, confocal, differential phase, fluorescence