A fluorometric viability assay for single human and rat islets

A microfluorometric viability assay for isolated human and rat islets of Langerhans has been developed using the fluorochromes fluorescein diacetate and propidium iodide. Fluorescein diacetate causes live cells to fluoresce green under blue light excitation (490 nm); propidium iodide causes dead cells to fluoresce red under green light excitation (454 nm). The fluorescence intensity from the live and dead cells within a single islet was selectively measured by photometry using 520 nm and 610 nm barrier filters with blue and green light excitation respectively. All measurements were corrected for background fluorescence. The proportion of dead cells within single human or rat islets measured by microfluorometry was found to correlate highly significantly (r = 0.99, P less than 0.001) with the proportion of dead cells measured by dissociating the same islet into a single cell suspension and counting the actual proportion of dead cells. This assay therefore provides a rapid, accurate and objective measurement of the proportion of dead cells within isolated human and rat islets.