TITLE: Attachment 1: Preparation of Fluorescein Diacetate and Propidium Iodide Stock Solutions  (FDA/PI)
AUTHORS: Integrated Islet Distribution Program, Carol J Swanson, Barbara Olack

[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]
?. [Preparation of Stock Fluorescein Diacetate (FDA) Solution]
Prepare  Fluorescein Diacetate (FDA) according to the formulation in sub-steps below.
The expiration date, for both PI and FDA stains, is six months from the date of preparation. Both of the fluorescent dyes used in this assay are light sensitive and must be kept in the dark, covered with aluminum foil.
?. [Preparation of Stock Propidium Iodide (PI)]
Prepare  Propidium Iodide (PI) according to the formulation in sub-steps below.
?. [Preparation of Stock Fluorescein Diacetate (FDA) Solution]
Dissolve 0.00199 g of FDA in 200 mL of acetone in a glass bottle and cover with aluminum foil.  FDA Supplier__________________________________________  FDA Lot # ____________________________________________Expiration Date _______________________________________  Acetone Supplier_______________________________________  Acetone Lot # _________________________________________Expiration Date ________________________________________Acetone Supplier_______________________________________  Acetone Lot # _________________________________________Expiration Date ________________________________________
[FDA]
[Acetone]
?. [Preparation of Stock Fluorescein Diacetate (FDA) Solution]
Calculation: FDA FW = 416.4  Stock Concentration = 24 µM   Volume required = 200 mLFormula:  FW X Concentration X Volume = (416.4) x (24 x 10-6) X (200 x 10-3) = 0.00199 g FDASigma Aldrich has a Mass Molarity Calculator that can be used to determine smaller quantities: https://www.sigmaaldrich.com/chemistry/stockroom-reagents/learning-center/technical-library/mass-molarity-calculator.html
?. [Preparation of Stock Fluorescein Diacetate (FDA) Solution]
Store Stock FDA solution tightly sealed in 4 mL or smaller aliquots in polypropylene tubes or glass tubes at ≤  , protected from light, for up to six months.          FDA (24 µM) Stock Solution Assigned Lot number:___________________________          Expiration Date: ____________________________________________________________          Prepared By/Date: _________________________________________________________          Reviewed By/Date: _________________________________________________________
-20 °C
?. [Preparation of Stock Propidium Iodide (PI)]
Dissolve 0.0125 g of PI in 25 mL of DPBS and cover with aluminum foil.  PI Supplier__________________________________________  PI Lot # ____________________________________________Expiration Date _____________________________________  PBS Supplier________________________________________  PBS Lot # ___________________________________________Expiration Date ______________________________________PBS Supplier________________________________________  PBS Lot # ___________________________________________Expiration Date ______________________________________
[Propidium Iodide]
[DPBS]
?. [Preparation of Stock Propidium Iodide (PI)]
Calculation: PI FW = 668.4  Stock Concentration = 750 µM  Volume required = 25 mLFW X Concentration X Volume = (668.4) x (750 x 10-6) X (25 x 10-3) = 0.0125 g PI
?. [Preparation of Stock Propidium Iodide (PI)]
Store Stock PI solution tightly sealed in 0.5 mL or smaller aliquots in polypropylene Microcentrifuge Snap Cap tubes or glass tubes at   to  , protected from light, for up to six months.          PI (750 µM) Stock Solution Assigned Lot number:___________________________          Expiration Date: ____________________________________________________________          Prepared By/Date: _________________________________________________________          Reviewed By/Date: _________________________________________________________
2 °C
8 °C