- Probe sonicator (Ultrasonic homogenizer).
- Insulin Ultra-sensitive kit (62IN2PEH, Cisbio).
- 384 wells, low volume plates (white).
3 Methods
3.1 Isolation
and Maintenance
of Pancreatic Islets
Isolation of Mouse Pancreatic Islets
To isolate mouse pancreatic islets, we use the method based in
collagenase injection into the pancreatic duct. This method has
been thoroughly detailed before by Carter and colleagues [20]
and ourselves [21]. Here we provide a brief description of the
protocol with some minor modifications that our lab currently
applies, which results in maximum yields of 250–300 islets per
mouse.- Prepare 3 (for C57/BL6 mice) to 5 (for CD1 mice) ml of
collagenase solution per mice. - Cull the animal by cervical dislocation following locally regu-
lated guidelines. - Wet fur with 70% (v/v) ethanol and open the abdomen to
expose the organs in the peritoneal cavity. - With the help of sharp scissors, cut the lower ribs and the
xiphoid cartilage. This will provide an adequate angle for nee-
dle insertion and will therefore facilitate the subsequent injec-
tion of collagenase inside the duct. - Position the animal under a Zoom stereomicroscope (10
objective), on top of a 15 ml tube transversally located under-
neath the liver area (also to provide a good angle for needle
insertion in the duct), tail pointing away from the researcher.
Flip the intestine toward the left-hand side. - Clamp the duct entry into the small intestine (seeNote1).
Leave the clamp toward the left-hand side of the mouse. Hold-
ing and directing the clamp with one hand while injecting with
the other one will aid the positioning of the needle inside
the duct. - Pull the liver down, which should expose the Y-like junction
between the ducts originating from the liver and gallbladder.
Using a 30G (for C57/BL6 mice) or 27G (for CD1 mice)
needle attached to a 5 ml syringe, cannulate and steadily inject
through the duct 3 (for C57/BL6 mice) to 5 (for CD1 mice)
ml of collagenase solution (seeNotes 2– 4 ). - Excise the inflated pancreas and place it inside a 50 ml tube on
ice until all the samples have been taken (seeNote 5).
Manipulation and Measurement in Islets 419