- 37°C water bath
- collagenase P (Roche store @ -20°C)
- HBSS (Gibco #14025, store @ 4°C
- 1 ml and 5 ml syringe
- 18G, 27G, and 30G needles
- 5.0 suture (Roboz sut-15-1 spool suture)
- cotton swab
- Nembutal (50 mg/ml)
- 70% EtOH
- 0.9% sterile saline (for diluting Nebutol)
- BD 12 ml round bottom tubes
- Warm water bath to 37°C
- Prepare collagenase P from 10X (12.5 mg/ml in HBSS + 0.2% BSA)
stock (stored @ -80°C) in HBSS at 1.25 mg/ml. Thaw 1 - 2
vials of stock solution, depending on how many mice will be
sacrificed. Each mouse requires 3.8-4 ml diluted
- Draw up ~3.5 ml of diluted collagenase solution into a 5ml
syringe with an 18G needle and place the syringe on ice.
Prepare (on ice) a separate 500 μl aliquot of the collagenase
solution for dissected pancreas.
- Bend the 30G needle under microscope to ~45° angel so that the
needle bevel is facing up.
- Ready supplies and solutions including: cotton swabs, bench
paper, suture, and 0.1% BSA HBSS (add 400 μl 12.5% BSA to 5O
- Inject mouse with 5 mg/ml Nembutal (20 μl/g BW) using a 1 ml
syringe with a 27G needle. After about after 5-10 minutes,
check to see that the mouse is fully anesthetized by pinching the
feet. If there is a response, inject an additional 500 μl of
Nembutal, and wait until there is no pinching response.
- Place the anesthetized mouse on its back, spread the legs and
gently tape them in an open position. Disinfect mouse abdomen
with 70% EtOH.
- Open the abdomen and locate the bile duct. Close the bile
duct at the duodenum by suture ligation.
- Remove the 18G needle from the collagenase syringe and replace
it with the bent 30G needle. Inject collagenase into bile
duct and inflate the pancreas.
- Cut suture close to the knot. Separate the pancreas from
the duodenum and stomach. Once exposed, remove the pancreas
with the spleen attached. Remove the spleen, blot pancreas on
a clean absorbent paper. Place the pancreas in a 12 ml tube
containing 500 μl collagenase solution and place on ice.
- Perform cervical dislocation on the mouse and discard the
Pancreas digestion and islet collection
- Incubate pancreata at 37°C for 18 - 20 min.
- Add ~ 5 ml cold HBSS and shake vigorously (~ 3 "shakes"), add
up to 12 ml HBSS, centrifuge for 1 min at 1000 rpm.
- Wash islets in 12 ml HBSS 3 times (same as #2 above)
- Add 8 ml HBSS to digested pancreas and pour the digested tissue
into a 10 cm plate; add an additional 4 ml HBSS to wash the tube
and pour the wash into the same plate.
- Hand pick islets under a stereoscope using a 100 μl
pipette. Transfer the picked islets to a new plate containing
HBSS, and then again to a third plate of HBSS using a 10 μl pipette
to pick up islets. Place the islets in a 1.5 ml eppendorf
tube. Keep the plates that contain the islets on ice when not
- Wash the islets once in 1.0 ml HBSS and carefully take out the
supernatant. Freeze the pellets that contain the islets @
-80°C until ready to perform future analysis.
Written by Yanyun Gu 8/07/2007