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Rapid assessment of islet viability with acridine orange and propidium iodide

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Summary

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.

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References

  1. Andersson, A.; Sandler, S. Viability tests of cryopreserved endocrine pancreatic cells. Cryobiology 20: 161–168; 1983.

    Article  PubMed  CAS  Google Scholar 

  2. Andersson, A. Isolated mouse pancreatic islets in culture: Effects of serum and different culture media on the insulin production of the islets. Diabetologia 14: 397–404; 1978.

    Article  PubMed  CAS  Google Scholar 

  3. Andersson, A.; Westman, J.; Hellerstrom, C. Effect of glucose on the ultrastructure and insulin biosynthesis of isolated mouse pancreatic islets maintained in tissue culture. Diabetologia 10: 743–753; 1974.

    Article  PubMed  CAS  Google Scholar 

  4. Bank, H. L.; Davis, R. F.; Emerson, D. Cryogenic preservation of isolated rat islets of Langerhans: Effect of cooling and warming rates. Diabetologia 16: 195–199; 1979.

    Article  PubMed  CAS  Google Scholar 

  5. Bank, H. L.; Reichard, L. Cryogenic preservation of isolated islets of Langerhans: Two-step cooling. Cryobiology 18: 489–496; 1981.

    Article  PubMed  CAS  Google Scholar 

  6. Cooperstein, S. J.; Watkins, D. The islets of Langerhans: biochemistry, physiology, and pathology. New York: Academic Press; 1981.

    Google Scholar 

  7. Corliss, D. A.; White, W. E., Jr. Fluorescence of yeast vitally stained with ethidium bromide and propidium iodide. J. Histochem. Cytochem. 29: 45–48; 1981.

    PubMed  CAS  Google Scholar 

  8. Darzynkiewicz, Z.; Evenson, D.; Kapuscinski, J., et al. Denaturation of RNA and DNA in situ induced by acridine orange. Exp. Cell Res. 148: 31–46; 1983.

    Article  PubMed  CAS  Google Scholar 

  9. Darzynkiewicz, Z.; Traganos, F.; Kapuscinski, J., et al. Accessibility of DNA in situ to various fluorochromes: Relationship to chromatin changes during erythroid differentiation of friend leukemia cells. Cytometry 5: 355–363; 1984.

    Article  PubMed  CAS  Google Scholar 

  10. Edidin, M.; Church, J. A. A quantitative fluorochromatic assay for alloantibodies. Transplantation 6: 1010–1014; 1968.

    Article  PubMed  CAS  Google Scholar 

  11. Edidin, M. A rapid, quantitative fluorescence assay for cell damage by cytotoxic antibodies. J. Immunol. 104: 1303–1306; 1970.

    PubMed  CAS  Google Scholar 

  12. Evans, H. M.; Schulemann, W. The action of vital stains belonging to the benzidine group. Science 34: 443–453; 1914.

    Article  Google Scholar 

  13. Golden, J. F.; West, S. S.; Echols, C. K., et al. Quantitative fluorescence spectrophotometry of acridine-orange unfixed cells: potential for automated detection of human uterine cancer. J. Histochem. Cytochem. 24: 315–321; 1976.

    PubMed  CAS  Google Scholar 

  14. Hedig, L. G. Determination of total serum insulin (IRI) in insulin-treated diabetic patients. Diabetologia 8: 260–266; 1972.

    Article  Google Scholar 

  15. Jones, K. H.; Senft, J. A. An improved method to determine cell viability by simultaneous staining with fluorescein diacetatepropidium iodide. J. Histochem. Cytochem. 33: 77–79; 1985.

    PubMed  CAS  Google Scholar 

  16. Kapuscinski, J.; Darzynkiewicz, Z.; Melamed, M. R. Interactions of acridine orange with nucleic acids. Properties of complexes of acridine orange with single stranded ribonucleic acid. Biochem. Pharmacol. 32: 3679–3694; 1983.

    Article  PubMed  CAS  Google Scholar 

  17. Krebs, A. T.; Gierlach, Z. S. Vital staining with the fluorochrome acridine orange and its application to radiobiology. Am. J. Roentgenol. Rad. Ther. 65: 93–96; 1951.

    CAS  Google Scholar 

  18. Krishan, A. Rapid flow cytofluormetric analysis of mammalian cells by propidium iodide staining. J. Cell. Biol. 66: 188–193; 1975.

    Article  PubMed  CAS  Google Scholar 

  19. Lacy, P. E.; Davie, J. M.; Finke, E. H. Transplantation of insulin-producing tissue. Am. J. Med. 70: 589–594; 1981.

    Article  PubMed  CAS  Google Scholar 

  20. Lacy, P. E.; Walker, M. M.; Fink, C. J. Perifusion of isolated rat islets in vitro: Participation of the microtubular system in the biphasic release of insulin. Diabetes 21: 987–988; 1972.

    PubMed  CAS  Google Scholar 

  21. Lacy, P. E.; Kostianovsky, M. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16: 35–39; 1967.

    PubMed  CAS  Google Scholar 

  22. Lerman, L. S. The structure of the DNA-acridine complex. Proc. Natl. Acad. Sci. USA 49: 94–102; 1963.

    Article  PubMed  CAS  Google Scholar 

  23. McKay, D. B.; Karow, A. M., Jr. Factors to consider in the assessment of viability of cryopreserved islets of Langerhans. Cryobiology 20: 151–160; 1982.

    Article  Google Scholar 

  24. Moore, P. L.; Didyk, R. D.; Bank, H. L. 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; 1985.

    Article  Google Scholar 

  25. Moore, P. L.; Didyk, R.; Bank, H. L. The interpretation of a new method for assessing islet viability with fluorescent dyes. Diabetes 35: 177a; 1986.

    Google Scholar 

  26. Nakamura, N.; Hurst, R. E.; West, S. S., et al. Biophysical cytochemical investigations of intracellular heparin in neoplastic mast cells. J. Histochem. Cytochem. 28: 223–230; 1980.

    PubMed  CAS  Google Scholar 

  27. Pace, C. S.; Sachs, G. Glucose-induced proton uptake in secretory granules of beta-cells in monolayer culture. Am. J. Physiol. 242: C382–387; 1982.

    PubMed  CAS  Google Scholar 

  28. Pantazis, C. G.; Kniker, W. T. Assessment of blood leukocyte microbial killing by using a new fluorochrome in microassay. J. Reticuloendothel. Soc. 26: 155–170; 1979.

    PubMed  CAS  Google Scholar 

  29. Persidsky, M. D.; Baillie, G. S. Fluorometric test of cell membrane integrity. Cryobiology 14: 322–331; 1977.

    Article  PubMed  CAS  Google Scholar 

  30. Prowse, S. J.; Lafferty, K. J.; Simeonovid, C. J., et al. The reversal of diabetes by pancreatic islet transplantation. Diabetes 31 (Suppl) 4: 30–38; 1982.

    PubMed  Google Scholar 

  31. Reaven, E. P.; Gold, G.; Reaven, G. Effect of age on glucose stimulated insulin release by the B-cell of the rat. J. Clin. Invest. 64: 591–599; 1982.

    Article  Google Scholar 

  32. Rotman, B.; Papermaster, B. W. Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc. Natl. Acad. Sci. USA 55: 134–141; 1966.

    Article  PubMed  CAS  Google Scholar 

  33. Sandler, S.; Andersson, A.; Swenne, I., et al. Structure and function of human fetal endocrine pancreas before and after cryopreservation. Cryobiology 20: 230–236; 1983.

    Article  PubMed  CAS  Google Scholar 

  34. Scharp, D. W.; Downing, R.; Merrell, R. C., et al. New approaches in the methods for isolating mammalian islets of Langerhans utilizing dog pancreas. In: Federlin, K.; Bretzel, R. G., eds. Islet isolation, culture, and cryopreservation. New York: Thiema-Stratton; 1981.

    Google Scholar 

  35. Strugger, S. Fluorescence microscope examination of trypanosomas in blood. Can. J. Res. Sect. E. 26: 229–231; 1948.

    Google Scholar 

  36. Sutherland, D. E. R. Pancreas and islet transplantation. I. Experimental studies. Diabetologia 20: 161–185; 1981.

    PubMed  CAS  Google Scholar 

  37. Taylor, M. J.; Duffy, T. J.; Hunt, C. J., et al. Transplantation andin vitro perifusion of rat islets of Langerhans after slow cooling and warming in the presence of either glycerol or dimethyl sulfoxide. Cryobiology 20: 185–204; 1983.

    Article  PubMed  CAS  Google Scholar 

  38. Traganos, F.; Adams, L. R.; Kamentsky, L. A., et al. Critical effect of the dye concentration on acridine orange fluorescence of fixed thymocytes. Acta Cytol 16: 281–283; 1972.

    PubMed  CAS  Google Scholar 

  39. Tyrer, H. W.; Golden, J. F.; Vansickel, M. H., et al. Automatic cell identification and enrichment in lung cancer. II. Acridine orange for cell sorting of sputum. J. Histochem. Cytochem. 27: 552–560; 1979.

    PubMed  CAS  Google Scholar 

  40. Waring, M. Ethidium and propidium. In: Corcoran, J. W.; Hahn, F. E., eds. Antibiotics III. New York: Springer-Verlag; 1975: 141–165.

    Google Scholar 

  41. Weill, G.; Calvin, M. Optical properties of chromophobeacromolecule complexes: absorption and fluorescence of acridine dyes bound to polyphosphates and DNA. Biopolymers 1: 401–417; 1963.

    Article  CAS  Google Scholar 

  42. Welsh, M.; Hellerstrom, C.; Andersson, A. Respiration and insulin release in mouse pancreatic islets. Effects ofl-leucine and 2-ketoisocaproate in combination withd-glucose andd-glutamine. Biochem. Biophys. Acta. 721: 178–190; 1982.

    Article  PubMed  CAS  Google Scholar 

  43. West, S. S. Fluorescence microspectrophotometry of supravitally-stained cells. In: Pollister, A. W., ed. Physical techniques in biological research, vol. 30, 2nd ed. New York: Academic Press; 1969: 253–321.

    Google Scholar 

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Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, 29425, Charleston, South Carolina

    Harvey L. Bank

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  1. Harvey L. Bank
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This research was supported in part by the National Institutes of Health, Bethesda, MD, grant DK 18115.

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Bank, H.L. Rapid assessment of islet viability with acridine orange and propidium iodide. In Vitro Cell Dev Biol 24, 266–273 (1988). https://doi.org/10.1007/BF02628826

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  • DOI: https://doi.org/10.1007/BF02628826

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