Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

Abstract

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories [1][2][3][4] , variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations 5,6 , but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh 7,8 , in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient 9 , and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a 6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate 6 . This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay 10 . Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

Divergent compensatory responses to high-fat diet between C57BL6/J and C57BLKS/J inbred mouse strains

“…Mouse islets were isolated from collagenase-perfused pancreata (CIzyme MA; VitaCyte, Indianapolis, IN) and neutral protease (CIzyme BP Protease, VitaCyte) as previously described (38). Glucose-stimulated insulin secretion (GSIS) was performed as previously described, using ϳ40 islets per mouse (15).…”

The Inactivation of RabGAP Function of AS160 Promotes Lysosomal Degradation of GLUT4 and Causes Postprandial Hyperglycemia and Hyperinsulinemia

“…Mouse islets were isolated as previously described (28). Glucose-stimulated insulin secretion assay was performed in islets with comparable sizes for 1 h. Supernatants were collected for insulin measurement using an ELISA kit (EZRMI-13K; EMD Millipore, Billerica, MA), and islets were lysed and used for immunoblotting analysis.…”

Transcriptional Activity of the Islet β Cell Factor Pdx1 Is Augmented by Lysine Methylation Catalyzed by the Methyltransferase Set7/9

“…Intraperitoneal glucose tolerance tests using 2 g/kg body weight glucose proceeded as described previously (22). Islets were isolated from collagenase-perfused pancreata as described previously (23). Static glucose-stimulated insulin secretion assays using islets were performed as described previously (22), and insulin released into the medium was normalized to total islet insulin content.…”