A methodological study of discontinuous Percoll-gradient separation of pancreatic islets from rats

Abstract

A discontinuous Percoll gradient was used to separate pancreatic islets from rats. Adjusting the osmolality to 315 mosmol kg^-1 H₂O, working at 4 °C and filtration of the pancreatic digest prior to gradient separation were vital for securing an optimal separation. The gradient design was also critical. Thus, optimal results were obtained with gradient centrifugation at 800 g, and using a bottom gradient that maintained the tissue evenly distributed prior to centrifugation. No significant difference was observed in the insulin release from islets isolated manually and islets isolated on Percoll gradients. Neither was there any significant differences in the insulin release from Percoll-separated islets stimulated in solutions containing 25 mmol 1^-1 NaHCO₃ and islets stimulated in solutions containing only 4.2 mmol 1^-1 NaHCO₃. In conclusion, discontinuous ^-Percoll gradients can be used for isolation of pancreatic islets from rats.

Key-words:

• anti-aggregation
• bicarbonate buffer
• insulin release
• islet yield
• osmolality
• temperature