TITLE: Viability Estimation of Islets for Distribution Using Inclusion and Exclusion Fluorescent Dyes (FDA/PI)
AUTHORS: Integrated Islet Distribution Program

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This Standard Operating Procedure is adapted from the work of the '</span><span style = "font-style:italic;">National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities'' </span><span>following the SOP cited in the document </span><span style = "font-style:italic;">'Purified Human Pancreatic Islet - Viability Estimation of Islet Using Fluorescent Dyes (FDA/PI): Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium'</span></div><div class = "text-block">This SOP defines the procedure for assessment of viability of human isolated islet preparations, which include endocrine and exocrine tissue, for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored research in the Integrated Islet Distribution Program (IIDP).  This protocol is written to assist the participating islet isolation centers and investigators who are part of this program.</div><div class = "text-block">Fluorescein Diacetate/ Propidium Iodide (FDA)/(PI) Viability Assay is a rapid fluorometric method to test the integrity of the plasma membrane simultaneously using inclusion and exclusion dyes; the assay differentiates between viable and nonviable cells and is, consequently, used for determination of viability of islet preparations.</div></div>

[STEPS]
?. [Islet Assessment Preparation]
Assemble all items described in the “Assay Set Up” section.
?. [Islet Assessment Preparation]
Prepare for Fluorescent Microscopy and Image Recording: Refer to the Microscope and camera using manufacture instructions and settings.
?. [Islet Assessment Preparation]
Prepare [IIDP Islet Viability Worksheet] for computer tally in Step 8 with calculations using pre-formatted programming.
?. [Preparation of Working Dye Solutions]
Obtain Stock Solutions prepared previously using the link to Attachment 1:       Attachment 1: Preparation of  Fluorescein Diacetate and Propidium Iodide Stock Solutions  (FDA/PI) Remove a 4 mL aliquot of   FDA stock solution from the freezer (≤ - )Verify that the expiration date for the FDA stock solution is not expired.  The expiration date is six months from the date of preparation.Attachment 1: Preparation of  Fluorescein Diacetate and Propidium Iodide Stock Solutions  (FDA/PI)Remove a 4 mL aliquot of  FDA stock solution from the freezer (≤ -)Record lot number and Expiration Date of the  FDA stock solution.            Stock Solution Lot#____________________________________            Stock Solution Expiration Date _________________________
Both of the fluorescent dyes used in this assay are light sensitive and must be kept in the dark, covered with aluminum foil.
-20 °C
?. [Preparation of Working Dye Solutions]
Remove a 0.5 mL aliquot of PI stock solution from the refrigerator ( to ).Record lot number and Expiration Date of the  PI stock solution.            Stock Solution Lot#____________________________________            Stock Solution Expiration Date _________________________
2 °C
8 °C
?. [Preparation of Islets for Viability Staining]
Prepare islets for viability assessment.  Obtain islet aliquot for viability assessment.
?. [Preparation of Islets for Viability Staining]
Add 460 µL DPBS to the culture dish in which islets will be dyed and counted.
This volume will adjust the final working concentrations of FDA to 0.46 Micromolar (µM) and PI to 14.34 Micromolar (µM).
?. [Preparation of Islets for Viability Staining]
Mix islet flask well and using a large orifice pipet tip, take an aliquot that would equal roughly 100 to 200 IEQ before islets settle and place it into a separate, 2 mL Snap Cap Microcentrifuge Tube.
?. [Preparation of Islets for Viability Staining]
Allow islets from the aliquot to settle for 2 to 3 minutes and, using a large orifice pipet tip, take 43 µL of settled islets from the bottom of the tube and add to the culture dish containing the 460 µL DPBS.
?. [Preparation of Islets for Viability Staining]
Quickly add first 10 µL of Stock PI and then 10 µL of Stock FDA to the islet suspension.  Gently swirl to mix. Turn off the lights in the room.
?. [Estimation and Calculation of Viability]
Assess the preparation immediately using the fluorescent microscope using either a green/red filter set or a single emission filter determined by which microscope is used.   FDA is a cell permeable esterase substrate. The fluorescein substrate readily diffuses into cells where intracellular esterases hydrolyze the dye to produce fluorescein-A negatively charged molecule, which diffuses out of intact cells very slowly. Fluorescein emits light energy at a wave length of 517 nm.PI is poorly permeable to living cells, but readily permeable to dead cells and binds to the DNA. When bound to the DNA, PI emits light energy at a wavelength of 617 nmSome microscopes use green/red filter set to detect each color under the appropriate filter, and superimpose the images to get the final image.  Note, however, filters allow one to observe the fluorescing as red alone, or red and green together, as the green fluorescence is much brighter than the red and can overshadow one's interpretation of the viability.Some microscopes use only a single emission filter to detect both green and red fluorescence so that both colors can be seen at the same time.PI is poorly permeable to living cells, but readily permeable to dead cells and binds to the DNA. When bound to the DNA, PI emits light energy at a wavelength of 617 nmSome microscopes use green/red filter set to detect each color under the appropriate filter, and superimpose the images to get the final image.  Note, however, filters allow one to observe the fluorescing as red alone, or red and green together, as the green fluorescence is much brighter than the red and can overshadow one's interpretation of the viability.Some microscopes use only a single emission filter to detect both green and red fluorescence so that both colors can be seen at the same time.Some microscopes use green/red filter set to detect each color under the appropriate filter, and superimpose the images to get the final image.  Note, however, filters allow one to observe the fluorescing as red alone, or red and green together, as the green fluorescence is much brighter than the red and can overshadow one's interpretation of the viability.Some microscopes use only a single emission filter to detect both green and red fluorescence so that both colors can be seen at the same time.
?. [Estimation and Calculation of Viability]
The assessment must take place as quickly as possible once the dye is added to the islets. 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.
?. [Estimation and Calculation of Viability]
Estimate the percentage of viable tissue vs. total tissue as follows:
Note:  FDA produces bright green fluorescence (viable cells) cells, while PI produces red Fluorescence (dead or dying cells). Islets are aggregates of cells, therefore, it is expected that some cells within an individual islet will stain green and some will stain red. The objective of the test is to assess, as accurately as possible, the percentage of islet cells that are viable.
?. [Estimation and Calculation of Viability]
Evaluate the viability of 50 consecutive tissue particles.
?. [Estimation and Calculation of Viability]
Determine the percent viability for each tissue particle by estimating to the nearest 1% the viable (green fluorescence) tissue quantity vs. the total (viable plus non-viable, green and red fluorescence) tissue quantity.Few or no cells are green; Average viability = non-viableLess than 33% of islet cells are green; Average viability = 25%Between 33 and 66 % of islet cells are green; Average viability = 50%Greater than 66% of the islet cells are green; Average viability = 75%Few or no cells are red; Average viability = 100%
?. [Estimation and Calculation of Viability]
After assessment, swirl contents of the petri dish to the center of the dish and capture a fluorescent image of the preparation and document with Isolation Number, Date, RRID, Sample Type (Post-Purification, Post-Culture, or Pre-shipping) as well as Purity Layer (High, Middle, or Low).
?. [Estimation and Calculation of Viability]
Tally results and record all data and calculations on the attached [IIDP Viability Worksheet] in this step.  Auto calculate the percent viability mean and standard deviation of the 50 observations made.Counted by ______________________________________  Date ________________________Approved by _____________________________________   Date _______________________
?. [Estimation and Calculation of Viability]
Discard unused stain, do not freeze.
?. [Interpretation of Results]
The 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.
?. [Interpretation of Results]
The PI stains the nuclei of dead/non-viable cells only.  Dead cells appear bight fluorescent red/orange.  PI does not cross the membrane of viable cells.