Isolation and Characterization of Mouse Islets

Islets were isolated from 8 to 12 weeks old C57BL/6 male mice as described before by our laboratory^{31 (link)–34 (link)}. Briefly, mouse islets were isolated using perfusion and digestion of pancreas with collagenase V (from Roche), density gradient purification with histopaque-1077 (Sigma), and then hand-picked. Isolated islets were cultured overnight in RPMI 1640 containing 10% FBS, 11 mM glucose, and then switched for 1 h to Krebs Ringer Bicarbonate buffer containing 2.6 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 4.9 mM KCl, 98.5 mM NaCl, and 25.9 mM NaHCO₃ (all from Sigma-Aldrich) supplemented with 20 mM Na-HEPES and 0.1% BSA. About 10 islets in each experimental condition were transferred to each well in 24-well plate containing 2.8 mM and 16.7 mM glucose concentration in Krebs Ringer Bicarbonate buffer for 1 h. The supernatants were collected for insulin measurements. The islets were lysed with 1% Triton to determine total protein content in the islets. Insulin levels were measured with an ELISA kit from ALPCO. About 200 isolated mouse islets from WT or sparc −/− mice were also collected for Western blot analysis of RGS4 and SPARC.