Summary
A rapid fluorometric 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.
Article PDF
Similar content being viewed by others
References
Edidin M (1970) A rapid, quantitative fluorescence assay for cell damage by cytotoxic antibodies. J Immunol 104: 1303–1306
Mohr LR, Trounson AO (1980) The use of fluorescein diacetate to assess embryo viability in the mouse. J Reprod Fertil 58: 189–196
Rotman B, Papermaster BW (1966) Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc Natl Acad Sci USA 55: 134–141
Bank HL (1980) Viability of frozen rat granulocytes and granulocyte precursors. Cryobiology 17: 262–272
Pantazis CG, Kniker WT (1979) Assessment of blood leukocyte microbial killing by using a new fluorochrome microassay. J Reticuloendothel Soc 26: 155–170
West SS (1969) Fluorescence microspectrophotometry of supravitally stained cells. In: Pollister AW (ed) Physical techniques in biological research, 2nd ed, Vol 3 c. Academic Press, New York, pp 253–321
Kapuscinski J, Darzynkiewicz Z, Melamed MR (1983) Interactions of acridine orange with nucleic acids. Properties of complexes of acridine orange with single stranded ribonucleic acid. Biochem Pharmacol 32: 3679–3694
Waring M (1975) Ethidium and propidium. In: Corcoran JW, Hahn FE (eds) Antibiotics III. Mechanism of action of antimicrobial and antitumor agents. Springer, New York, pp 141–165
Yeh C-JG, Hsi B-L, Faulk WP (1981) Propidium iodide as a nuclear marker in immunofluorescence. II. Use with cellular identification and viability studies. J Immunol Methods 43: 269–275
Tanke HJ, Van der Linden PWG, Langerak J (1982) Alternative fluorochromes to ethidium bromide for automated read out of cytotoxicity tests. J Immunol Methods 52: 91–96
Jacobs DB, Pipho C (1983) Use of propidium iodide staining and flow cytometry to measure antibody-mediated cytotoxicity: resolution of complement-sensitive and resistant target cells. J Immunol Methods 62: 101–108
Moore PL, Didyk R, Bank HL (1985) The rapid assessment of viability of isolated islets of Langerhans with fluorescent dyes: relationship between fluorescent images and insulin content. J Cell Biol 101: 247a
Lacy PE, Walker MM, Fink CJ (1972) Perifusion of isolated rat islets in vitro: participation of the microtubular system in the biphasic release of insulin. Diabetes 21: 987–998
Bank HL, Davis RF, Emerson D (1979) Cryogenic preservation of isolated rat islets of Langerhans: effect of cooling and warming rates. Diabetologia 16: 195–199
Gray DW, McShane P, Grant A, Morris PJ (1984) A method for isolation of islets of Langerhans from the human pancreas. Diabetes 33: 1055–1061
Gotoh M, Maki T, Kiyoizumi T, Satomi T, Monaco AP (1985) An improved method for isolation of mouse pancreatic islets. Transplantation 40: 437–438
Horaguchi A, Merrell RC (1981) Preparation of viable islet cells from dogs by a new method. Diabetes 30: 455–458
Hutz RJ, DeMayo FJ, Dukelow WR (1985) The use of vital dyes to assess embryonic viability in the hamster, Mesocricetus Auratu. Stain Technol 60: 163–167
Dolan MF (1965) Viability assays — a critique. Fed Proc 24 [Suppl]: S275–279
Klebe RJ (1984) Rapid cloning of mammalian cells with honeycomb cloning plates and nonlethal vital stains. In Vitro 20: 127–132
Bank HL (1988) The rapid assessment of islet viability with Acridine orange and propidium iodide. In Vitro 24 (in press)
Taylor MJ, Bank HL, Benton MJ (1987) Selective destruction of leukocytes by freezing as a potential means of modifying tissue immunogenicity: membrane integrity of Lymphocytes and Macrophages. Cryobiology 24: 91–102
Albright BL, Selinger DS, Reed WP (1979) Use of vital stains to study bacterial adherence to epithelial cells. Stain Technol 54: 347–349
Black L, Berenbaum MC (1964) Factors affecting the dye exclusion test for cell viability. Exp Cell Res 35: 9–13
Tennant JR (1964) Evaluation of the trypan blue technique for determination of cell viability. Transplantation 2: 685–694
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bank, H.L. Assessment of islet cell viability using fluorescent dyes. Diabetologia 30, 812–816 (1987). https://doi.org/10.1007/BF00275748
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00275748