TITLE:  Qualitative Assessment of Islet Viability by Staining with Fluorescein Diacetate (FDA) and Propidium Iodide (PI) Dyes
AUTHORS: Human Islet Phenotyping Program (HIPP) of the IIDP

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;font-weight:bold;">This Standard Operating Procedure (SOP) is adapted from the Viability SOP from the Clinical Islet Transplant Consortium Protocol and the Integrated Islet Distribution Program (IIDP) for Distribution Centers. This SOP provides the Human Islet Phenotyping Program (HIPP) procedures for Fluorescein Diacetate (FDA) and Propidium Iodide (PI) staining and imaging of islet preparation for qualitative viability assessment.</span></div><div class = "text-block">This SOP defines the assay method used by the Human Islet Phenotyping Program (HIPP) for quantitative and qualitative determination of the Purified Human Pancreatic Islet product, post-shipment, manufactured for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)-sponsored research in the Integrated Islet Distribution Program (IIDP). </div></div>

[STEPS]
?. [Procedures]
Limitations
?. [Procedures]
Once the dye is added to the islets, the assessment must take place  as quickly as possible. If there is a delay of more than 15 minutes, the  accuracy of the assessment will be diminished as the islets lose their  viability with time.
?. [Procedures]
Both of the fluorescent dyes used in this assay are light sensitive and must be kept in the dark, covered with aluminum foil.
?. [Procedures]
The fluorescent dyes are temperature sensitive and must be stored as follows:  FDA: ≤ -20ºCPI: 2 - 8ºC
?. [Procedures]
Assay Set Up
?. [Procedures]
Assemble all items described in the “Supplies and Materials” section.
?. [Procedures]
Prepare Fluorescent dyes: Fluorescein Diacetate (FDA) and Propidium Iodide (PI), if required.
?. [Procedures]
Preparation of Fluorescein Diacetate and Propidium Iodide
?. [Procedures]
Remove FDA from the freezer and PI from the refrigerator. Weigh the required amount of the reagent on an analytical balance.
?. [Procedures]
Dissolve  of FDA in  of acetone in a glass bottle and cover with aluminum foil. Store in  aliquots at -20°C.
0.00199 g
200 mL
10 mL
?. [Procedures]
Dissolve  of PI in  of DPBS and cover with aluminum foil. Store in  aliquots at 2-8ºC.
0.0125 g
25 mL
5 mL
?. [Procedures]
Discard used stain.
?. [Procedures]
Record the expiration date on each aliquot tube. The expiration date, for both PI and FDA stains, is six months from the date of preparation.
?. [Procedures]
Staining and Estimation of Viability
?. [Procedures]
Add two  replicates of well-mixed total islet suspension to the culture dish.
400 µl
?. [Procedures]
Quickly add first of PI and then of FDA to the islet suspension. Gently swirl to mix.
10 µl
10 µl
?. [Procedures]
Turn off the lights in the room.
?. [Procedures]
Incubate plate in the dark for 15 minutes.
?. [Procedures]
Assess the preparation immediately using the fluorescent microscope.  Image islets using an Olympus SZX12 stereomicroscope system. Capture  multiple fields ensuring that FDA/PI staining is visualized in 50 – 100  islets per preparation.
FDA produces bright green fluorescence in viable cells, while PI produces red fluorescence in dead or dying cells.
?. [Procedures]
Interpretation of Results
?. [Procedures]
FDA freely passes through the cell membrane of live cells. Viable  cells appear bright fluorescent green when stained with FDA. In a live  cell, FDA is hydrolyzed to the polar free fluorescein, and it is trapped  within the intact membranes of the viable cells present in islets.
?. [Procedures]
PI stains the nuclei of dead/non-viable cells only. Dead cells appear  bright fluorescent red/orange. PI does not cross the membrane of viable  cells.
?. [Procedures]
FDA/PI staining captures viable and dead cells in both islets and  residual exocrine tissue. This staining provides a rapid qualitative  viability assessment of the human islet preparation; however, the 3-D  nature of islets and exocrine tissue remnants precludes accurate cell  counting. The Vanderbilt HIPP will supplement viability assessment with  counts of live and dead cells in a single cell suspension using Countess  II technology (Life Technologies AMQAX1000) (see HIPP Protocol-04-v02 Quantitative Assessment of Islet Viability by Trypan Blue Staining).
?. [Data Storage and Reporting]
To facilitate data management and ensure data security, the Vanderbilt HIPP uses an institutional server-based platform for data storage and analysis.
?. [Data Storage and Reporting]
Annotated images containing metadata (Figure 1) will be uploaded to  the IIDP-HIPP database and immediately disseminated to IIDP-affiliated  investigators and islet isolation centers.
?. [Deviations and Resolutions:]
Document any deviations that occurred from this protocol that affect  the final results and report with the analysis of the assay.