A simple, rapid method for estimating the viability of isolated islets of Langerhans with fluorescent dyes is described. Low concentrations of acridine orange and propidium iodide (AO/PI) were used to visualize living and dead islet cells simultaneously. AO/PI-stained islets can be divided into three distinct groups. Group A islets fluoresce green, contain insulin, and have normal ultrastructure; group C islets fluoresce primarily red, contain little or no insulin, and have cells with disrupted cellular membranes. Group B islets fluoresce red, green, and yellow. The yellow color is due to the addition of two primary colors from the superimposed red and green fluorescing cells. In this assay, the interpretation that red islet cells are dead and green islet cells are alive was confirmed by sequentially staining single islet cells with AO/PI and trypan blue. The observation that red islets are dead was confirmed by heat-killing, enzymatically damaging, treating with ethanol, or depriving islets of nutrients and observing the red fluorescence. This assay should be useful in studies where the assessment of islet viability is essential.

Summary.A rapid fluorometfic method has been developed to evaluate the viability of isolated islet cells. The assay differentiates between viable and nonviable cells by the simultaneous use of the inclusion and exclusion dyes acridine orange and propidium iodide. When viewed by fluorescent microscopy, viable cells fluoresce green, while nonviable cells fluoresce bright red. Although the acridine orange and propidium iodide assay measures membrane integrity, the results of this assay correlate with other measures of cell viability. Compared to trypan blue exclusion, this assay is easier to read, more stable, and has fewer staining artifacts. The assay enables the rapid estimation of the viability of a population of islet cells prior to time-consuming experiments rather than retrospectively. This assay can also be used with intact islets. Stained islets can be divided into three distinct groups: green fluorescing islets contain insulin, red fluorescing islets contain little or no insulin and a third class of islets containing some non-viable cells fluoresce red, green, and yellow. The yellow colour is due to the superimposition of red and green fluorescing cells. Key words:Islet viability, fluorochromes, B cells, acridine orange, propidium iodide, trypan blue exclusion, membrane integrity.A reliable method for rapidly determining the viability of isolated islet cells would be useful in studies on transplantation, cryopreservation, insulin release, and biosynthesis. Functional assays to determine insulin content or release and morphological assays including histochemistry or electron microscopy are time-consuming and expensive for routine viability assays.An alternative approach uses inclusion and/or exclusion dyes to test the integrity of the plasma membrane. Several fluorometric dyes were tested as indicators of membrane integrity. Fluorescein diacetate (FDA) has been used as a fluorometric assay of cell viability. FDA is a nonpolar ester which passes through plasma membranes and is hydrolysed by intracellular esterases to produce free fluorescein. The polar fluorescein is confined within cells with an intact plasma membrane and can be observed under appropriate excitation conditions [1][2][3][4]. However, staining of islet cells with FDA results in high background fluorescence. This fluorescence is presumably due to extracellular hydrolysis of the FDA or to fluorescein that leaked through damaged membranes.Acridine orange (AO) is a membrane-permeable, monovalent, cationic dye [5,6] which binds to nucleic acids. A low concentration of AO causes a green fluorescence, while a high concentration causes a red fluorescence. Propidium iodide (PI) is impermeable to intact plasma membranes, but it easily penetrates the plasma membrane of dead or dying cells [7] and intercalates with DNA or RNA forming a bright red fluorescent complex [8][9][10][11]. Both intracellular and extracellular background fluorescence is minimal after staining with optimal concentrations of these dyes.A combination of low concentrations of AO and...

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