Ti e base

Islets were transduced with adenovirus harboring the FRET sensor, Epac2-camps^{56 (link)} (a kind gift from Prof. Dermot Cooper, University of Cambridge), before imaging using a Crest X-Light spinning disk system coupled to a Nikon Ti-E base and ×10/0.4 NA objective. Excitation was delivered at λ = 430–450 nm using a Lumencor Spectra X light engine. Emitted signals were detected at λ = 460–500 and λ = 520–550 nm for Cerulean and Citrine, respectively, using a Photometrics Delta Evolve EM-CCD. Imaging was performed in HEPES–bicarbonate buffer, containing (in mmol/L) 120 NaCl, 4.8 KCl, 24 NaHCO₃, 0.5 Na₂HPO₄, 5 HEPES, 2.5 CaCl₂, 1.2 MgCl₂, and 3–17 d-glucose. Vehicle (H₂O), Exendin4(1–39) (10–20 nM), or Liraglutide (10 nM) were applied at the indicated time points, with forskolin (10 µM) acting as a positive control.

Islets were hand-picked after collagenase digestion of the whole pancreas [23 ], and cultured (5% CO₂, 37°C) in RPMI medium containing 10% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin. Islets were loaded with Fluo-8 (AAT Bioquest, USA; Cat#21082) dissolved in DMSO containing 20% pluronic acid. Islets were then imaged using an X-Light spinning disk system (Crest, Italy) coupled to a Ti-E base (Nikon, Japan) and ×10/0.4/air objective (Nikon, Japan). Excitation was delivered at λ = 458–482 nm using a Spectra X-light engine (Lumencor, USA), with emitted signals detected at λ = 500–550 nm using a Delta Evolve EM-CCD (Teledyne Photometrics, USA). Imaging buffer contained (in mmol/l) 120 NaCl, 4.8 KCl, 24 NaHCO₃, 0.5 Na₂HPO₄, 5 HEPES, 2.5 CaCl₂, 1.2 MgCl₂, and 3–17 d-glucose. Data were analysed using ImageJ, with traces presented as F/F_min where F = fluorescence at any timepoint and F_min = minimum fluorescence. No data were excluded unless the cells displayed a clear non-physiological state (i.e. impaired viability).