TITLE: Mouse Pancreatic Islet Isolation
AUTHORS: Nancy Smith, Aliya Spigelman, Haopeng Lin, Patrick Macdonald

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol details islet isolation from mouse pancreas. The protocol is divided into 3 main parts; </span><span style = "font-style:italic;">in situ </span><span>perfusion of pancreas with collagenase, pancreas digestion, and islet purification.  A link is provided for the purification using histopque gradient.</span></div></div>

[STEPS]
?. [Solution Prep- Hanks’ Balanced Salts (HBSS) - Sigma H6136]
Measure out 900ml of room temperature H2O.
?. [Solution Prep- Hanks’ Balanced Salts (HBSS) - Sigma H6136]
While gently stirring the water, add the powdered medium. Stir until dissolved. DO NOT HEAT.
?. [Solution Prep- Hanks’ Balanced Salts (HBSS) - Sigma H6136]
Rinse original vial with water to remove traces and add to above.
?. [Solution Prep- Hanks’ Balanced Salts (HBSS) - Sigma H6136]
Add 0.35g sodium bicarbonate and stir until dissolved.
[Sodium Bicarbonate]
?. [Solution Prep- Hanks’ Balanced Salts (HBSS) - Sigma H6136]
Adjust pH to 7.4
?. [Solution Prep- Hanks’ Balanced Salts (HBSS) - Sigma H6136]
Bring solution to 1L.
?. [Solution Prep- Hanks’ Balanced Salts (HBSS) - Sigma H6136]
Store at 4°C
4 °C
?. [Solution Prep- Collagenase type V – Sigma C9263 lot 010M8620:]
Dissolve 1mg/ml Collagenase in HBSS from above.Approximately 5ml per mouse for injection and shakingMake fresh before isolation, and keep on ice (use within the hour)
?. [Digestion]
After isolation, allow digestion to proceed for 7 minutes (lot specific -Sigma C9263 lot 010M8620 ) in shaking water bath at 37°C.
37 °C
?. [Digestion]
Shake digested material vigorously.
?. [Digestion]
Add Mouse Islet Media to stop digestion.  Tubes remain on ice until ready for purification.
?. [Purification]
Allow islets to settle (2-3 minutes) and remove excess fat.  Proceed to the histopaque gradient protocol to purify the islets.  If hand picking is preferred, go to the next step.https://www.protocols.io/view/purification-of-mouse-pancreatic-islets-using-hist-u7ueznw
?. [Purification]
Pour islets into a non tissue culture dish and pick islets into a 35mm non tissue culture dish.
?. [Purification]
Once islets picked cleanly, culture using Mouse Islet Media in 37°C, 5% CO2 until ready for use in experiment.
?. [Pancreas Perfusion]
Euthanize mouse according to your institute's research ethics protocols. (We use a CO2 chamber)
?. [Pancreas Perfusion]
Common bile duct is tied or clamped where it meets the intestine.
?. [Pancreas Perfusion]
Collagenase is injected in situ via the common bile duct using a size 30G needle until the pancreas is inflated (typically 1-2 mL). The head and the tail of the pancreas should be inflated to maximize the number of islets isolated.
?. [Pancreas Perfusion]
The pancreas is removed and placed in tube (15mL or 50mL depends on preference)  with remaining collagenase (1-2mL) and kept on ice until the next step.
?. [Pancreas Perfusion]
Make a midline incision from the lower abdomen to the sternum.
?. [Soultion Prep - Mouse Islet Culture Media]
Add FBS and Pen/Strep to bottle of RPMI  AB1500ml RPMI 1640 (11.1mM glucose)Gibco 11875-119250ml FBS Canadian Origin Gibco 12483-02035ml Pen/strep (10000 Unit/ml/10000 ug/ml)Gibco 15140-122
AB1500ml RPMI 1640 (11.1mM glucose)Gibco 11875-119250ml FBS Canadian Origin Gibco 12483-02035ml Pen/strep (10000 Unit/ml/10000 ug/ml)Gibco 15140-122
?. [Soultion Prep - Mouse Islet Culture Media]
Store at 4°C
4 °C