Isolation and Enrichment of Pancreatic Islet Cells

 

  1. Anaesthetise mice by intraperitoneal injection of approximately 0.1mg xylazine hydrochloride (Rhomazine 20; Jurox , Australia ) and 0.3mg ketamine hydrochloride (Ketalar; Parke-Davis , Australia ) in PBS per 10g bodyweight
  2. Excise skin covering the belly to expose the peritoneal muscle layer
  3. Make an incision in the peritoneal muscle layer to expose internal organs
  4. Bleed out mice via perforation of the heart
  5. Using a dissecting microscope locate the common bile duct and clamp at the duodenum
  6. Make a small incision in the duct using micro-scissors and insert a 30g needle
  7. Infuse the Pancreas with 3ml of solution A (3mg/ml Collagenase P, 15mg/ml BSA, 0.01mg/ml DNase and 20mM HEPES in Hanks buffered solution).
  8. Excise the Pancreas and place into 1ml of fresh solution A and incubate at 37 oC for 13mins
  9. Vortex the digest and place on ice for 5mins
  10. Remove supernatant and add 1ml of fresh solution A, followed by another 5 minute incubation on ice
  11. Again remove the supernatant and add 1ml of solution B (10% BSA, 20mM HEPES in HANKS).
  12. After another 5 minute incubation on ice, remove the supernatant and replace with 1ml of solution B
  13. Pick Islets from the remaining exocrine tissue under a dissecting microscope and place into media A on ice.
  14. Transfer Islets to 1ml of Trypsin-EDTA solution for 4 minutes.
  15. Use a blunt 20g needle to draw up and expel the islets to mechanically disrupt them
  16. Resuspend cells in 2mL of HANKS
  17. Perform viable cell counts using a haemocytometer and fluorescent microscope after ethidium bromide/acridine orange staining, or using a Z2 Coulter Counter (Beckman Coulter USA ), with viable cells gated by size