title: Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans.
abstract: BACKGROUND: A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO(R)13, SYTO(R)24 and SYBR(R)14 as possible alternatives to FDA. RESULTS: We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO(R)13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. CONCLUSIONS: From a review of the literature and from our observations on the impact of reagent handling and various staining and imaging parameters used to visually evaluate islets, consistent interpretation of islet cell membrane integrity and viability is dependent upon a number of factors. We discuss the utility and limitations of these reagents in evaluating islet cell membrane integrity and viability.
authors.affiliation: Diabetes Institute for Immunology and Transplantation, University of Minnesota, 425 Harvard, Minneapolis, MN 55455.
authors.forename: Vinc
authors.initials: V
authors.lastname: Boyd
authors.forename: Olivia Maria
authors.initials: OM
authors.lastname: Cholewa
authors.forename: Klearchos K
authors.initials: KK
authors.lastname: Papas
references.citation: Artif Organs. 2003 Nov;27(11):1053-6
references.pmid: 14616525
references.citation: Horm Metab Res Suppl. 1990;25:82-7
references.pmid: 2088991
references.citation: Cryobiology. 1996 Apr;33(2):236-52
references.pmid: 8674356
references.citation: Med J Aust. 2006 Mar 6;184(5):221-5
references.pmid: 16515432
references.citation: Cell Transplant. 2000 Sep-Oct;9(5):681-6
references.pmid: 11144965
references.citation: Pancreas. 2000 Apr;20(3):270-6
references.pmid: 10766453
references.citation: J Endocrinol. 1999 Jun;161(3):357-64
references.pmid: 10333538
references.citation: Am J Transplant. 2005 Jul;5(7):1635-45
references.pmid: 15943621
references.citation: Transplant Proc. 2005 Oct;37(8):3501-4
references.pmid: 16298642
references.citation: Cell Transplant. 2004;13(5):481-8
references.pmid: 15565860
references.citation: Horm Metab Res. 1985 Aug;17(8):391-5
references.pmid: 3932174
references.citation: J Microsc. 1993 Mar;169(Pt 3):329-38
references.pmid: 8478913
references.citation: Transplant Proc. 2004 Jun;36(5):1613-4
references.pmid: 15251397
references.citation: Diabetes. 1989 Jan;38 Suppl 1:185-8
references.pmid: 2492002
references.citation: Surgery. 1999 Aug;126(2):393-8
references.pmid: 10455912
references.citation: J Endocrinol. 1999 Nov;163(2):181-90
references.pmid: 10556766
references.citation: Diabetes. 1967 Jan;16(1):35-9
references.pmid: 5333500
references.citation: Hum Immunol. 1999 Dec;60(12):1303-9
references.pmid: 10626746
references.citation: In Vitro Cell Dev Biol. 1991 Aug;27A(8):633-8
references.pmid: 1917780
references.citation: Transplantation. 2005 Apr 15;79(7):842-5
references.pmid: 15818328
references.citation: Diabetes. 2006 Feb;55(2):312-7
references.pmid: 16443762
references.citation: Biomed Sci Instrum. 1993;29:111-9
references.pmid: 8329581
references.citation: Biotechnol Bioeng. 1998 Mar 20;57(6):655-65
references.pmid: 10099245
references.citation: Transplant Proc. 2005 Oct;37(8):3412-4
references.pmid: 16298611
references.citation: Am J Transplant. 2007 Jan;7(1):38-47
references.pmid: 17227556
references.citation: J Histochem Cytochem. 2001 Apr;49(4):519-28
references.pmid: 11259455
references.citation: Diabetes Res. 1989 Nov;12(3):141-9
references.pmid: 2699586
references.citation: Am J Transplant. 2007 Mar;7(3):707-13
references.pmid: 17229069
references.citation: Transplant Proc. 2004 Jun;36(5):1528-31
references.pmid: 15251377
references.citation: Methods Cell Biol. 1989;30:1-498
references.pmid: 2927326
references.citation: In Vitro Cell Dev Biol. 1988 Apr;24(4):266-73
references.pmid: 3284875
references.citation: Surgery. 1999 Aug;126(2):299-304
references.pmid: 10455898
references.citation: Stain Technol. 1987 Nov;62(6):373-81
references.pmid: 2448922
references.citation: Am J Transplant. 2003 Jan;3(1):53-63
references.pmid: 12492711
references.citation: Res Commun Chem Pathol Pharmacol. 1984 Apr;44(1):83-92
references.pmid: 6427864
references.citation: J Control Release. 2005 Sep 20;107(1):65-77
references.pmid: 15993507
references.citation: Am J Transplant. 2007 Apr;7(4):1010-20
references.pmid: 17391141
references.citation: J Cell Biol. 1973 Dec;59(3):766-71
references.pmid: 4128323
references.citation: Am J Transplant. 2005 Nov;5(11):2671-81
references.pmid: 16212626
references.citation: Diabetologia. 1987 Oct;30(10):812-6
references.pmid: 3322907
references.citation: Acta Diabetol Lat. 1990 Jul-Sep;27(3):185-95
references.pmid: 2075782
references.citation: Cell Transplant. 2005;14(10):757-62
references.pmid: 16454350
references.citation: Surgery. 2004 Aug;136(2):336-45
references.pmid: 15300200
references.citation: Transplant Proc. 2001 Feb-Mar;33(1-2):1709
references.pmid: 11267479
references.citation: Ann N Y Acad Sci. 2001 Nov;944:96-119
references.pmid: 11797699
references.citation: Biochem Biophys Res Commun. 2000 Oct 22;277(2):455-61
references.pmid: 11032744
references.citation: Nat Med. 2006 Mar;12(3):301-3
references.pmid: 16491083
references.citation: Transplantation. 2006 May 27;81(10):1471-2; author reply 1472
references.pmid: 16732188
references.citation: N Engl J Med. 2000 Jul 27;343(4):230-8
references.pmid: 10911004
references.citation: Histochem J. 1976 Jul;8(4):341-50
references.pmid: 60305
references.citation: Am J Transplant. 2004 Mar;4(3):390-401
references.pmid: 14961992
references.citation: Am J Transplant. 2005 Jan;5(1):21-30
references.pmid: 15636608
references.citation: Transplantation. 2007 Jan 15;83(1):7-12
references.pmid: 17220782
references.citation: Proc Natl Acad Sci U S A. 1966 Jan;55(1):134-41
references.pmid: 5220862
references.citation: Endocrinology. 1976 Feb;98(2):429-37
references.pmid: 765121
references.citation: Transplant Proc. 1997 Feb-Mar;29(1-2):750-2
references.pmid: 9123509
references.citation: Expert Opin Biol Ther. 2004 Mar;4(3):269-77
references.pmid: 15006723