title: From the rat to the beta cell: a fast and effective technique of separation of Langerhans islets and direct purification of pancreatic beta cells.
abstract: Isolated Langerhans islets represent a useful model for the study of the endocrine pancreas. The possibility to purify pancreatic beta cells from a mixed Langerhans islet cell population may lead towards a dedicated focus on beta cell research. We describe an effective and rapid immunomagnetic technique for the direct purification of beta cells from isolated Langerhans islets of rat. After the sacrifice of the rat, the Langerhans islets were separated by ductal injection of the pancreas with collagenase, altered to a mixed Langerhans islet cell population and incubated with conditioned immunomagnetic beads targeted to the beta cell surface. The beads were previously coated with a specific antibody against the surface of the beta cell, namely K14D10. The suspension of mixed Langerhans islet cells and immunomagnetic K14D10-conditioned beads was pelleted by a magnetic particle concentrator to isolate the bead-bound cells, which were finally suspended in a culture medium. The purified cells were immunoreactive for insulin and no glucagon-positive cells were detected at immunocytochemistry. Real Time PCR confirmed the purification of the pancreatic beta cells. This immunomagnetic technique allows a rapid, effective and consistent purification of beta cells from isolated Langerhans islets in a direct manner by conditioning the immunomagnetic beads only. This technique is easy, fast and reproducible. It promises to be a reliable method for providing purified beta cells for in vitro research.
authors.affiliation: Department of Endocrinology and Diabetes Mellitus, St Vincent's University Hospital, University College Dublin , Ireland and.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Tamagno
authors.forename: Simonetta
authors.initials: S
authors.lastname: Vigolo
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Olivieri
authors.forename: Chiara
authors.initials: C
authors.lastname: Martini
authors.forename: Eugenio
authors.initials: E
authors.lastname: De Carlo
mesh_terms: D000818:Animals; D007150:Immunohistochemistry / Q000379:methods*; D050417:Insulin-Secreting Cells / Q000737:chemistry*; D007515:Islets of Langerhans / Q000737:chemistry*; D008297:Male; D051381:Rats; D017208:Rats, Wistar