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

Order of Publishing in Issue: 
7
Volume :2
Issue :2
March, 2008
Page No: 
66-84
Authors: 
Vinc Boyd1, Olivia Maria Cholewa2*, Klearchos K. Papas1
Address: 
1Diabetes Institute for Immunology and Transplantation, University of Minnesota, 425 Harvard, Minneapolis, MN 55455, USA
Address: 
2Molecular Probes, Inc., 29851 Willow Creek Road, Eugene, OR 97402, USA

A review of current literature shows that the combined use of the cell permeable esterasesubstratefluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide(PI) to be one of the most common fluorescencebased methods to assess the viability of isolatedislets of Langerhans, and it is currently used for islet product release prior to transplantation inhumans. However, results from this assay do not correlate with islet viability and function or islettransplantation success in animals or humans (20, 21). This may be in part attributed to considerabledifferences as well as discrepancies in the use of these reagents on islets. We critically surveyedthe literature and evaluated the impact of a number of variables associated with the use ofFDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated otherfluorescent stains, such as SYTO®13, SYTO®24 and SYBR®14 as possible alternatives to FDA.It was 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 ofexamining islets were significant in the final scoring of viability. For archival file photos, theexposure time and camera/software settings can also impact interpretation of viability. Althoughour results show that FDA does detect intracellular esterase activity and staining withPI 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 nudemice. In addition, the use of two nucleic acid stains, such as SYTO®13 and PI, for live/deadscoring exhibited staining anomalies which limit their accuracy in assessing islet viability. Froma review of the literature and from our observations on the impact of reagent handlingand various staining and imaging parameters used to visually evaluate islets, consistentinterpretation 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 membraneintegrity and viability.

Keywords: 
Fluorescein Diacetate, Propidium Iodide, Viability, Islets of Langerhans.
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