Islet Protocol

Digestive enzyme liberase:
“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 2 Bottles
72ul DNase1 144ul DNase1
1.5ml HEPES 3ml HEPES
70ml HBSS 140ml HBSS

Make quenching buffer:

1 Bottle 2 Bottles
110ul DNase1 220ul Dnase1
11ml FBS 22ml FBS
100ml HBSS 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:
6 minutes
1,100 RPM
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: 
20 minutes
2,400 RPM
Slow acceleration/no deceleration
17.  Reset centrifuge temperature to 4°C.
18.  Separate media, islets, a pellet into three separate tubes.
19.  Centrifuge down:
6 minutes
1,100 RPM
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