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College of Life Sciences Nutrition, Dietetics, and Food Science
Tessem Laboratory - Diabetes Research Group

Islet Protocol

Digestive enzyme liberase:
Q`op9=\\\\\\\AQZ
“1 bottle = 6 rate at weening (2ml in each bottle)
Add 1ml ddH20/container and let sit on ice for 30
minutes
Add DNase1, HEPES, and HBSS and let sit on ice for 20 minutes

1 Bottle

72ul DNase1
1.5ml HEPES
70ml HBSS

2 Bottles

144ul DNase1
3ml HEPES
140ml HBSS

Make quenching buffer:

1 Bottle

110ul DNase1
11ml FBS
100ml HBSS

2 Bottles

220ul Dnase1
22ml FBS
200ml HBSS

Make histopaque 1100 by mixing:

  • 100ml histopaque 1077
  • 120 ml histopaque 1119
  1. Collect 1 conical tube per rat. Wet all conical tubes with liberase.
  2. Fill each conical tube with 5ml of liberase
  3. Dissect rats by profusing pancreas with 5ml liberase.
  4. Spread remaining liberase between all tubes. Be sure they are balanced.
  5. Place tubes in a 37°C water bath for 28 minutes
  6. Shake tubes in water bath every 2-3 minutes
  7. Remove tubes from water bath and shake each for 30 seconds
  8. Add 10 ml of Quenching buffer to each tube
  9. Funnel each tube using a metal mesh into a new tube
  10. Rinse all tubes with Quenching buffer and filter to get any leftover islets. Pour excess into tube. Be sure to balance tubes. You can add more Quenching butter to balance if needed
  11. Centrifuge down:
    1. 6 minutes
    2. 1,100 RPM
    3. 4°C
    4. Fast acceleration/Fast deceleration
  12. Reset centrifuge to increase temperature to 20°C
  13. Pour off supernate and keep pellet. Let tubes dry by placing on paper towel.
  14. Add 10 ml histopaque
  15. Slowly add 5 ml RPMI. Adding RPMI slowly will make an interface.
  16. Centrifuge down:
    1. 20 minutes
    2. 2,400 RPM
    3. 20°C
    4. Slow acceleration/no deceleration
  17. Reset centrifuge temperature to 4°C.
  18. Separate media, islets, a pellet into three separate tubes.
  19. Centrifuge down:
    1. 6 minutes
    2. 1,100 RPM
    3. 4°C
    4. Fast acceleration/fast deceleration
  20. Pour off islets to about 10ml. Keep excess. Re-suspend pellet and pour into petri dish for selection
  21. Re-suspend pellet and pour into petri dish
  22. Keep media and pour into petri dish
  23. Look at all part under the dissecting microscope and pick out islet. Place islets into 12-well plate with islet media.
  24. Treat cells with 1ul of desired virus