Diabetologia (1987) 30:812-816 Diabetologia Springer-Verlag 1987 Assessment of islet cell viability using fluorescent dyes H. L. Bank Department of Pathologyand LaboratoryMedicine, Medical Universityof South Carolina, Charleston, South Carolina, USA Summary. A rapid fluorometfic method has been developed of islet cells prior to time-consuming experiments rather than to evaluate the viability of isolated islet cells. The assay dif- retrospectively. This assay can also be used with intact islets. ferentiates between viable and nonviable cells by the simulta- Stained islets can be divided into three distinct groups: green neous use of the inclusion and exclusion dyes acridine fluorescing islets contain insulin, red fluorescing islets con- orange and propidium iodide. When viewed by fluorescent tain little or no insulin and a third class of islets containing microscopy, viable cells fluoresce green, while nonviable some non-viable cells fluoresce red, green, and yellow. The cells fluoresce bright red. Although the acridine orange and yellow colour is due to the superimposition of red and green propidium iodide assay measures membrane integrity, the re- fluorescing cells. sults of this assay correlate with other measures of cell viabil- ity. Compared to trypan blue exclusion, this assay is easier to Key words: Islet viability, fluorochromes, B cells, acridine read, more stable, and has fewer staining artifacts. The assay orange, propidium iodide, trypan blue exclusion, membrane enables the rapid estimation of the viability of a population integrity. A reliable method for rapidly determining the viability rescence. Propidium iodide (PI) is impermeable to in- of isolated islet cells would be useful in studies on tact plasma membranes, but it easily penetrates the transplantation, cryopreservation, insulin release, and plasma membrane of dead or dying cells [7] and inter- biosynthesis. Functional assays to determine insulin calates with DNA or RNA forming a bright red fluo- content or release and morphological assays including rescent complex [8-11]. Both intracellular and extracel- histochemistry or electron microscopy are time-con- lular background fluorescence is minimal after staining suming and expensive for routine viability assays. with optimal concentrations of these dyes. An alternative approach uses inclusion and/or ex- A combination of low concentrations of AO and PI clusion dyes to test the integrity of the plasma mem- was evaluated as a viability assay for islet cell suspen- brane. Several fluorometric dyes were tested as indica- sions. This combination of fluorochromes permits the tors of membrane integrity. Fluorescein diacetate simultaneous observation of cells with intact and with (FDA) has been used as a fluorometric assay of cell compromised plasma membranes [12]. However, any viability. FDA is a nonpolar ester which passes new assays must be compared with classical assays of through plasma membranes and is hydrolysed by in- cell "viability". In this report the AO/PI assay is com- tracellular esterases to produce free fluorescein. The pared to the traditional trypan blue exclusion assay. polar fluorescein is confined within cells with an intact The stability, cytotoxicity, and reproducibility of the as- plasma membrane and can be observed under appro- say was evaluated on isolated islet cells derived from priate excitation conditions [1-4]. However, staining of adult rat. In addition, the assay was applied to islet islet cells with FDA results in high background fluo- cells derived from mice, dogs and neonatal humans rescence. This fluorescence is presumably due to extra- and to isolated intact rat islets. cellular hydrolysis of the FDA or to fluorescein that leaked through damaged membranes. Acridine orange (AO) is a membrane-permeable, Materials and methods monovalent, cationic dye [5, 6] which binds to nucleic All National Institute of Health and institutional Animal Research acids. A low concentration of AO causes a green fluo- Committee guidelines for the welfare of research animals were fol- rescence, while a high concentration causes a red fluo- lowed. Long Evans Hooded (LEH) male rats (220-250 g) were fast- H. L. Bank: Fluorescent islet cell viability assay 813 ed for 12 to 18 h before the isolation of the islets. Pancreatic islets of Table 1. Effect of fluorochrome concentrations on rat islet cells Langerhans were isolated using the collagenase isolation procedure described previously [13, 14]. A single step, 11% Ficoll (LSM, Bionet- Concentration Fluorescence ics Lab, Litton, Kensington, Md, USA) separation was used to sepa- [gmol/l] AO a pI b rate islets from the tissue digest [14]. The islets were collected at the buffer/ficoll interface, and washed with phosphate buffered saline Background Cells Background Cells (PBS). 0.67 clear green clear no uptake Rat islets were dissociated with gentle and repeated pipetting or 1.5 clear green clear no uptake with additional collagenase and incubation for 3-5 rain at 37 ~ fol- 10 clear green clear no uptake lowed by gentle pipetting. Adult mouse, rat and canine islets as well 20 clear red clear no uptake as human neisidioblastic neonatal islets were isolated according to 40 green red clear no uptake previously described methods [15-17] and dissociated as described 90 green red clear no uptake above. 190 green red clear no uptake Isolated islet cells were placed in PBS buffer containing 0.2% 390 green red clear no uptake (w/v) trypan blue (Gibco, Grand Island, NY, USA) or a combina- 780 green red clear no uptake tion of AO (Sigma, St. Louis, Mo, USA) and PI (Sigma) in PBS buf- 1.560 green red red red fer. Stained cells were examined using a 10X/0.4 NA Wild Fluotar 3.130 green red red red phase contrast lens on an inverted Wild M40 microscope equipped 6.250 green red red red for phase contrast and epi-illumination fluorescent microscopy. An 12.500 green red red red FITC fiker set (Ex490 : Em510) permitted the simultaneous visualiza- a Acridine Orange (AO) enters live cells and fluoresces green at low tion of green and red fluorescing cells. dye concentrations. At concentrations >__20 pomol/l, the intracellular Since high concentrations of many dyes are toxic to cells, a serial fluorescence undergoes a metachromatic shift from green to red. At dilution of AO and PI was made over the concentration range of AO concentrations >40 lxmol/1, an intense green extracellular back- 0.67-12,500 p~mol/1 and the fluorochromes were tested on freshly ground obscured the fluorescent cells, b Propidium Iodide (PI) isolated rat islet cells to determine the useful concentration range for stains the nuclei of dead and dying cells bright red. At concentra- each fluoroehrome. Low concentrations of AO (0.67 p.mol/1) and PI tions > 1,560 Ixmol/1, both background and cellular staining was an (75 ~mol/l) were used for the AO/PI assay because they resulted in intense red. At concentrations <780 Ixmol/1, viable cells excluded a bright cellular fluorescence while minimizing extracellular fluores- dye and the background fluorescence was minimal cence. Once the optimal staining conditions for AO/PI were deter- mined, this assay was compared to the trypan blue dye exclusion as- Results say performed under "optimal conditions". Dissociated rat islet cells were stained with AO/PI for 60 min and the percentage of cells which fluoresced green were compared with the percentage of the Table 1 shows the effects of varying either acridine same population of cells which excluded trypan blue after 5 rain. orange or propidium iodide concentrations on intracel- To determine if the dyes were toxic, dissociated rat islet cells lular and extracellular fluorescence in rat islet cell were exposed to either AO/PI or trypan blue for 60 and 5 rain, and preparations. When these cells were exposed to >__ the mean viabilities were compared. After exposure to the fluoro- 20 txmol/1 AO, an intracellular metachromatic shift chromes, the cells were placed in the dark at room temperature. Cell from green to red fluorescence was observed. At AO counts of trypan blue or AO/PI stained islet cells were made after 5 and 60 rain. At least 100 cells were counted per replicate, and a mini- concentrations >__40 [xmol/l islet cells were more diffi- mum of 3 replicates were counted per experiment to determine the cult to recognise against a bright green background of mean percent viable cells. extracellular fluorescence. At a concentration of To determine if the AO/PI assay could be applied to islet cells 0.67 gmol/l AO, islet cells fluoresced a bright green isolated from other species, dissociated cells from adult mouse, adult colour with very little extracellular fluorescence. dog, and human neonatal islets were observed after staining with When high concentrations of propidium iodide AO/PI or trypan blue for 5 or 60 min. were used, alone, (>_ 1560 ~tmol/1) resulted in a bright To assay the viability of intact islets, individual islets were placed red extracellular fluorescence (Table 1). At lower dye into separate wells of a 96 well microtiter plate, and stained with AO/PI. Staining time and dye concentration was identical to that de- concentrations extracellular fluorescence was progres- scribed above. Each islet was classified according to its fluorescent sively reduced, and fluorescent cells were discernable. colours. The criteria used for islet selection were: type A (> 90% At a concentration of 75 p.mol/1 PI [12], dead and dy- green), type B (40-60% green), or type C (>90% red). To confirm ing islet cells fluoresced a bright red colour with a low the interpretation that live islet ceils fluoresce green, and dead islet cells fluoresce red, dissociated islet ceils were stained for 5 min with extracellular fluorescence. AO/PI and photographed with fluorescence microscopy. Next the Table 2 compares AO/PI and trypan blue staining islets were washed free of AO/PI, stained for 5 min with trypan of dissociated rat islet cells.