Assessment of islet cell viability using fluorescent dyes

Abstract

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...

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method

“…The AO/PI staining procedure was performed using concentrations at 10 and 190 lM as previously described (Bank 1987). Fifty microliters of each naturally-dying cell sample at 0,6,12,24,48,72,96, and 168 h was mixed with 50 ll of PI, AO/PI, or 0.4 % TB (Sigma-Aldrich, St. Louis, MO, USA), and analyzed immediately with Cellometer Vision, AutoT4, and hemacytometer.…”

Effective glycemic control achieved by transplanting non-viral cationic liposome-mediated VEGF-transfected islets in streptozotocin-induced diabetic mice

“…To assess cell viability indirectly, we carried out 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetra-zolium bromide (MTT) assays as described previously (Bank, 1987) and acridine orange (0.67 µmol/L) and propidium iodide (75 µmol/L) (AO/PI) staining to visualize living and dead islet cells simultaneously. To assess the insulin secretory function of mice islets after transfection with effectene/pJDK or effectene/pJDK-VEGF complexes, transfection media were removed by centrifugation at 1,500 rpm and isolated islets were re-incubated at 37 o C for 1 h in KRBB solution containing low (3.33 mmol/L, basal) or high (16.65 mmol/L, stimulated) glucose concentrations.…”

Co-localization of nestin and insulin and expression of islet cell markers in long-term human pancreatic nestin-positive cell cultures

“…Islet-enriched fractions were pooled, stained with dithizone and quantified by counting under the microscope with the help of an optical net and then converted to the standard number of islet equivalents (IEQ), which represents the number of 150 µm diameter islets present in the sample. Islet cell viability, assessed by a fluorimetric cell viability assay (Live/Dead) according to Bank (1987), was usually greater than 80%. The purity of each preparation was assessed by comparing the amount of dithizone-stained endocrine tissue with the unstained exocrine (acinar) tissue.…”