The complete methodology for evaluating binding energies is described in the computational methods section

nm on a LSM 5Live microscope with a Plan-Apochromat 20x/0.8 NA lens. Images were collected at 1 frame every 2 Oleandrin site seconds to measure the fast oscillations in i generated by changes in ion channel conductances. Cells were imaged at 10 mM glucose for,510 min to allow sufficient time for synchronous oscillations to appear; the GPCR ligand and/or Gbc modulator of interest then was added and oscillations were continuously recorded for another,10 min. Data were normalized to the untreated control frequency or amplitude for each islet prior to addition of a GPCR ligand and/or Gbc modulator. Materials and Methods Islet Isolation All murine procedures were approved by and conducted in compliance with the Vanderbilt University Institutional Animal Care and Use Committee PubMed ID: operating under Public Health Service Animal Welfare Assurance A3227-01. All surgery procedures were performed following intraperitoneal injection of ketamine/xylazine anesthesia. Islet isolation protocol was performed as previously described. Briefly, pancreata from random healthy 812 week old C57BL/6 adult male mice were excised and digested in 0.150.22% Collagenase P per ml of Hank’s Balanced Salt Solution for 812 minutes under gentle agitation. Samples were centrifuged 3x and the supernatant was replaced with fresh HBSS. Individual islets were pipetted into fresh islet media and incubated overnight prior to use at 37uC and 5% CO2 for 2448 hours. Static Incubation Insulin Secretion Assays Islets were isolated as described above and allowed to recover overnight. Islets were pre-incubated for 1 hour in KRBH buffer consisting of 128.8 NaCl, 4.8 KCl, 1.2 KH2PO4, 1.2 MgSO4N7 H2O, 2.5 CaCl2, 20 Hepes, 5 NaHCO3 with 2.8 mM glucose. 4 islets per sample were incubated in 1 mL KRBH buffer at 2.8 mM, 10 mM, or 16.7 mM glucose with and without KP, GLP-1, gallein, or mSIRK individually or in combination for 45 minutes at 37uC; each condition was measured in triplicate. The samples were briefly spun at 3000 rpm and 500 mL of each sample were placed in a new tube. 500 mL of 2% Triton-X were added to each islet sample to lyse the islets prior to storage at 220uC. Insulin content and secretion were analyzed in duplicate with a Mouse Ultrasensitive Insulin ELISA kit and detected on a Spectra Max M5 spectrometer. Islet Dispersion Islets were isolated as described above and allowed to recover overnight. Islets were washed in DPBS and dispersed using Accutase for 10 minutes at 37uC under gentle agitation. Cells were centrifuged, washed with fresh islet media, and plated on glass bottom micro-well dishes. Plated cells were incubated prior to use at 37uC and 5% CO2 for 2448 hours. Microfluidic Device Construction Microfluidic devices were constructed using Sylgard 184 Silicone Elastomer base and curing agent mix that was degassed and cured on a master mold for 3 hours at 75uC. A well and two access holes for loading and removing the islets were created. The molds were bonded onto 22640 mm cover glass following plasma cleaning. Analysis and Statistics Data were analyzed with Microsoft Excel, ImageJ, MatLab, or GraphPad Prism software. For all imaging data, the background signal was subtracted and the mean 6 S.E. was determined. Student’s t-tests and ANOVAs were used where applicable and Welch’s PubMed ID: correction was used in cases where variance was statistically different between groups. p,0.05 was considered statistically significant unless otherwise noted. NADH Imaging All imaging experiments were conducted in