Isolation and Enrichment of Pancreatic Islet Cells
- 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
- Excise skin covering the belly to expose the peritoneal muscle layer
- Make an incision in the peritoneal muscle layer to expose internal organs
- Bleed out mice via perforation of the heart
- Using a dissecting microscope locate the common bile duct and clamp at the duodenum
- Make a small incision in the duct using micro-scissors and insert a 30g needle
- 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).
- Excise the Pancreas and place into 1ml of fresh solution A and incubate at 37 oC for 13mins
- Vortex the digest and place on ice for 5mins
- Remove supernatant and
add 1ml of fresh solution A, followed by another 5 minute incubation on ice
remove the supernatant and add 1ml of solution B (10% BSA, 20mM HEPES in HANKS).
- After another 5 minute incubation on ice, remove the supernatant and replace with 1ml of solution B
- Pick Islets from the remaining exocrine tissue under a dissecting microscope and place into media A on ice.
- Transfer Islets to 1ml of Trypsin-EDTA solution for 4 minutes.
- Use a blunt 20g needle to draw up and expel the islets to mechanically disrupt them
- Resuspend cells in 2mL of HANKS
- 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