TITLE: Human Islet Isolation Media Preparation
AUTHORS: James Lyon, Aliya Spigelman, Jocelyn E Manning Fox, Patrick Macdonald

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the preparation of various media formulations required for the isolation and culture of human islets, as performed by the Alberta Diabetes Institute IsletCore. </span><a href="http://www.bcell.org/adi-isletcore.html" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://www.bcell.org/adi-isletcore.html</span></a></div></div>

[STEPS]
?. [ HBSS (perfusion, rinse, priming solution), M199 (wash solution) and EMEM (dilution solution)]
ABCDE1Hank's Balanced Salt Solution - HBSS (perfusion, rinse, priming solutions) (10L)2ReagentConcentrationWeight/VolumeSupplierCatalogue #3HBSS Powder9.51 g/L1 bottleMediatech/Corning55-022-PB4CaCl2 (anhydrous)3.60 mM4.0gMP Biochemicals LLC153502905MgSO4 (anhydrous)0.81 mM0.98gVWR Life Science97061-4386NaHCO34.2 mM3.5gFisher ScientificS233-5007HEPES10mM23.83gFisher ScientificBP310-18Penicillin/Streptomycin100 U/ml penicillin 100 μg/ml streptomycin50mlLonza09-757F  ABCDE1Medium 199 - M199 (Wash media) (10L)2ReagentConcentrationWeight/volumeSupplierCatalogue #3M199 powder9.41g/L1 bottleMediatech/Corning90050PB4NaHCO326 mM22.0gFisher ScientificS233-5005HEPES10 mM23.83gFisher ScientificBP310-16Penicillin/Streptomycin100 U/ml penicillin 100 μg/ml streptomycin50mlLonza09-757F  ABCDE1Eagle Modified Minimal Essential Medium - EMEM (Dilution Media) (10L)2ReagentConcentrationWeight/volumeSupplierCatalogue #3EMEM powder9.23g/L1 bottleMediatech/Corning90009BP4NaHCO326 mM22.0gFisher ScientificS233-5005HEPES10 mM23.83gFisher ScientificBP310-16Penicillin/Streptomycin100 U/ml penicillin      100 μg/ml streptomycin50mlLonza09-757F
ABCDE1Hank's Balanced Salt Solution - HBSS (perfusion, rinse, priming solutions) (10L)2ReagentConcentrationWeight/VolumeSupplierCatalogue #3HBSS Powder9.51 g/L1 bottleMediatech/Corning55-022-PB4CaCl2 (anhydrous)3.60 mM4.0gMP Biochemicals LLC153502905MgSO4 (anhydrous)0.81 mM0.98gVWR Life Science97061-4386NaHCO34.2 mM3.5gFisher ScientificS233-5007HEPES10mM23.83gFisher ScientificBP310-18Penicillin/Streptomycin100 U/ml penicillin 100 μg/ml streptomycin50mlLonza09-757F
ABCDE1Medium 199 - M199 (Wash media) (10L)2ReagentConcentrationWeight/volumeSupplierCatalogue #3M199 powder9.41g/L1 bottleMediatech/Corning90050PB4NaHCO326 mM22.0gFisher ScientificS233-5005HEPES10 mM23.83gFisher ScientificBP310-16Penicillin/Streptomycin100 U/ml penicillin 100 μg/ml streptomycin50mlLonza09-757F
ABCDE1Eagle Modified Minimal Essential Medium - EMEM (Dilution Media) (10L)2ReagentConcentrationWeight/volumeSupplierCatalogue #3EMEM powder9.23g/L1 bottleMediatech/Corning90009BP4NaHCO326 mM22.0gFisher ScientificS233-5005HEPES10 mM23.83gFisher ScientificBP310-16Penicillin/Streptomycin100 U/ml penicillin      100 μg/ml streptomycin50mlLonza09-757F
?. [Prepare and pH the HBSS,  M199 and EMEM solutions]
Prepare the HBSS, M199, and EMEM solutions outlined in step 1 of the Stock Media Preparation table using the following directions:Dispense   of Milli-Q (18mΩ) water in to the carboy Store overnight at   to allow to come to temperature.Using the stirrer add the media powder to the water and allow to go into solution.Add the powdered supplements and Penicillin/Streptomycin to the appropriate media based on the above tables and allow to stir into solution.Stir the solution for   Store the prepared solution overnight at   to allow all powders to go into solution Stir the solution for   Calibrate the pH meter using the pH control buffersAdjust the pH level to   using the NaOH and/or HCl solutions.Bring to volume with the appropriate amount of Milli-Q water.Dispense  of Milli-Q (18mΩ) water in to the carboy Store overnight at  to allow to come to temperature.Using the stirrer add the media powder to the water and allow to go into solution.Add the powdered supplements and Penicillin/Streptomycin to the appropriate media based on the above tables and allow to stir into solution.Stir the solution for   Store the prepared solution overnight at   to allow all powders to go into solution Stir the solution for   Calibrate the pH meter using the pH control buffersAdjust the pH level to   using the NaOH and/or HCl solutions.Bring to volume with the appropriate amount of Milli-Q water.Stir the solution for Store the prepared solution overnight at  to allow all powders to go into solution Stir the solution for Calibrate the pH meter using the pH control buffersAdjust the pH level to   using the NaOH and/or HCl solutions.Bring to volume with the appropriate amount of Milli-Q water.Adjust the pH level to  using the NaOH and/or HCl solutions.
9 L
4 °C
4 °C
?. [Filter sterilize the HBSS, M199 and EMEM solutions]
Store the filtered media at  until needed for supplementation prior to isolation.
4 °C
?. [Prepare, pH and filter sterilize the 1M Nicotinamide solution]
ABCDE1Nicotinamide Solution - 1M (1L)2ReagentConcentrationWeight/volumeSupplierCatalogue #3Nicotinamide powder1 M122.12Sigma Aldrich Canada CoN06364HEPES10 mM2.383gFisher ScientificBP310-1
ABCDE1Nicotinamide Solution - 1M (1L)2ReagentConcentrationWeight/volumeSupplierCatalogue #3Nicotinamide powder1 M122.12Sigma Aldrich Canada CoN06364HEPES10 mM2.383gFisher ScientificBP310-1
?. [Prepare, pH and filter sterilize the 1M Nicotinamide solution]
Dispense  of Milli-Q water in to the carboy. Store overnight at  to allow to come to room temperature. Using the stirrer add the reagent powder into the water based on the table (Step 4) and allow to stir into solution.Stir the solution for   Calibrate the pH meter using the pH control buffersAdjust the pH level to   using the NaOH and/or HCl solutionsBring to volume (1L) with the appropriate amount of Milli-Q water. Filter sterilize using a bottle top filter (0.22μM) into a sterile 1L bottle.Stir the solution for Calibrate the pH meter using the pH control buffersAdjust the pH level to   using the NaOH and/or HCl solutionsBring to volume (1L) with the appropriate amount of Milli-Q water. Filter sterilize using a bottle top filter (0.22μM) into a sterile 1L bottle.Adjust the pH level to  using the NaOH and/or HCl solutionsBring to volume (1L) with the appropriate amount of Milli-Q water. Filter sterilize using a bottle top filter (0.22μM) into a sterile 1L bottle.
0.9 L
4 °C
?. [Preparation and use of Dithizone stain in Islet Preparations.]
Preparation of dithizoneWeigh out  of dithizone powder into a 50ml conical tube. Add  of DMSO and mix until the powder is in solution. Bring the resulting dithizone solution to 40ml total volume with HBSS and mix. Transfer the dithizone solution to a 60cc syringe with a 0.45µm nylon filter. UseFor every ml of islet suspension add an equal amount of the prepared dithizone solution must be added to the sample. UseFor visualization of staining add another  of HBSS to dilute the stain and reduce the background colour.Alternately,  of islet suspension,  dithizone and  HBSS.
0.2 g
6 mL
2 mL
100 µl
100 µl
200 µl
?. [Preparation of working solutions to be done the day of isolation.]
To prepare the working solution supplement each of the listed stock medias with the indicated supplements.  ABCDEFGH1Total volume per bottleBSA 30%&nbsp;HBSSEMEMM199Nicotinamide (1M)Betadine2(ml)(ml)(ml)(ml)(ml)(ml)(ml)3SOLUTION4M199 (aliquot into 12 x 100ml)1172160
  (4.0%)----------1000&nbsp;12-----5Dilution - make 3 bottles2020----------2000-----20-----6Priming solution1000-----1000--------------------7Wash 1 - split into 2x 550ml110595
  (2.5%)----------100010-----8Wash 2 - make 5 bottles105545 (1.125%)----------100010-----9Cannulation500-----500--------------------10Perfusion Solution350-----350--------------------11Decon 1300-----250---------------5012Decon 2250-----250--------------------13Decon 3250-----250--------------------  ABCDEFG1Gradient
  Collection tubes      2tube #1234563ml of wash 2 solution1001502002002252254tube #7891011125ml of wash 2 solution225225225225225150
ABCDEFGH1Total volume per bottleBSA 30%&nbsp;HBSSEMEMM199Nicotinamide (1M)Betadine2(ml)(ml)(ml)(ml)(ml)(ml)(ml)3SOLUTION4M199 (aliquot into 12 x 100ml)1172160
  (4.0%)----------1000&nbsp;12-----5Dilution - make 3 bottles2020----------2000-----20-----6Priming solution1000-----1000--------------------7Wash 1 - split into 2x 550ml110595
  (2.5%)----------100010-----8Wash 2 - make 5 bottles105545 (1.125%)----------100010-----9Cannulation500-----500--------------------10Perfusion Solution350-----350--------------------11Decon 1300-----250---------------5012Decon 2250-----250--------------------13Decon 3250-----250--------------------
ABCDEFG1Gradient
  Collection tubes      2tube #1234563ml of wash 2 solution1001502002002252254tube #7891011125ml of wash 2 solution225225225225225150
?. [Human Islet Culture media]
Preparation of the human islet culture mediaPrepare the culture media by supplementing CMRL by the following table.  Following these additions to the culture media, filter sterilize the resulting solution through a 0.22 µm  Stericup Sterile Vacuum Filtration System. CMRL 1066Bovine serum albumin 30%Insulin-Transferrin-Selenium (100x)Gibco® GlutaMAX Supplement Penicillin-Streptomycin Mixture(ml)(ml)(ml)(ml)(ml)Culture media5008.55102.5
CMRL 1066Bovine serum albumin 30%Insulin-Transferrin-Selenium (100x)Gibco® GlutaMAX Supplement Penicillin-Streptomycin Mixture(ml)(ml)(ml)(ml)(ml)Culture media5008.55102.5