Isolation of rodent pancreatic β cells
1. Adult rats weighing 250-350g were anesthetized, sacrificed and immediately used for pancreas sampling.
2. Rat islets were isolated from male wistar rats by digestion of primary cell solution.
3. The pancreas were excised and placed in a digestion of primary cell solution.
4. Tissue was minced and incubated for 40 minutes at 37°C.
5. After purification on a Ficoll gradient (Sigma), the isolated islets were washed twice in a phosphate-buffered saline (PBS) and the islets were cultured in primary cell system with 10% fetal calf serum.
6. The isolated islets were then exposed for 6-7 minutes at 37°C to the same medium supplemented with digestion of primary cell solution.
7. The incubation was stopped by the addition of primary cell solution, supplemented with 0.5% bovine serum albumin, 2.8 mM glucose and 10 mM Hepes, pH 7.4.
8. The resulting suspension, which comprised mostly single cells, was centrifuged for 5 minutes at 130g.
9. The pellet was re-suspended in primary cell system to obtain a final concentration of 3×105 cells.
10. For mouse islet isolation, pancreases were excised and placed in a primary cell system.
11. Tissue was minced and incubated for 38 minutes at 37°C.
12. After washing of the digested tissue, islets were hand picked under a stereomicroscope.
13. Rodent islets were cultured in primary cell system supplemented with 10% fetal calf serum.
14. The insulin-secreting cells (INS) were cultured primary cell system supplemented with 10% fetal calf serum, penicillin and streptomycin.
15. INS were kept at 37°C in a humidified incubator gassed with air and CO2 to maintain medium pH at 7.4, fed at 3 days intervals and passed by trypsinization once a week.
References:
1. Haefliger Jacques-Antoine, Tawadros Thomas, Meylan Laure, Gurun SabineLe, Roehrich Marc-Estienne, Martin David, Thorens Bernard, and Waeber Gerard. The scaffold protein IB1/JIP-1 is a critical mediator of cytokine-induced apoptosis in pancreatic beta cells, J Cell Sci. 2003: 116, 1463.
2. Asfari M, Janjic D, Meda P, Li G, Halban PA, and Wollheim CB. Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology. 1992; 130: 167.
