Murine Pancreatic Islet Isolation

Protocol

Collagenase Preparation

1. Weigh out the collagenase into a 50 ml conical and add isolation buffer to make the final concentration, as indicated in the table below. Dissolve the collagenase by vortexing.
   
   
   (12 mice + 2 extra mice = 14 mice x 5 ml/mouse = 70 ml total collagenase solution)
   
   
   (70 ml x 0.5 mg/ml or 0.3 mg/ml = 35 mg or 21 mg of collagenase P total)
   
   
   

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dithizone	reagent			Dissolve 5mg Dithizone in 5 ml of DMSO in a 50 ml conical tube. Add 45 ml of normal saline and mix gently. Filter with a 60 cc syringe and a 0.22 "m syringe filter into a new 50 ml tube. Store at 4 OC, until use. Add 1 ml to each 35 mm counting Grid-plate, when needed.
PBS	Buffer
HBSS	Buffer
Isolation soln.	Buffer			HBSS containing 10 mM of HEPES, 1 mM of MgCl2, 5 mM of Glucose, pH 7.4
Wash soln.	Buffer			Isolation buffer containing 1mM CaCl2
Collagenase P		Boehringer Ingeheim	1213873
Dissection tools				Hemostat clamp (n=2), Forceps (n=2), Scissors (for skin incision an for taking out pancreas from adjacent tissue)
Syringe				sterile, 5ml, 1/mouse.
30 G needles				sterile, 1/syringe
siliconized glass vials w/Teflon cap		Fisher Scientific	213-018-54	sterile, 1/mouse
large sink strainer				sterile
1000 ml Beaker				sterile
Gauze pads				4x4, 2/mouse
plastic eye dropper				sterile
Mice	Animal