Exendin 4

Isolated islet cell clusters were washed twice with DMEM/F-12 (Biological Industries), counted and mixed with Matrigel in glass centrifuge tubes. In all, 30,000–50,000 cells or 10–20 cell clusters were used per well of a pre-warmed 24-well plate (the volume of 50 µL). After Matrigel was solidified for 30 min at 37 °C, pancreatic islet expansion medium (PIEM) was added. PIEM consists of DMEM/F-12 (1% GlutaMax, 1% Penicillin-Streptomycin) plus 15% RSPO1 conditioned medium (home-made), 3 µM CHIR-99021 (BioGems), 1 µM A83-01 (Adooq Bioscience), 10 nM gastrin-1 (MedChemExpress), 1.25 mM N-acetylcysteine (Sigma), 10 µM Y-27632 (Adooq Bioscience), 10 µM FSK (TargetMol), 50 ng/mL Exendin-4 (ChinaPeptides), 50 µg/mL L-Ascorbic acid (Sigma), 250 nM 5-Iodotubercidin (Adooq Bioscience), 50 ng/mL FGF10 (PeproTech), 50 ng/mL EGF (PeproTech), 10 mM Nicotinamide (Sigma), B27 Supplement (minus Vitamin A) (Thermo). Cultures were kept at 37 °C, 5% CO₂ in a humidified incubator. During culturing, medium was refreshed every three days.

To assess the specificity of ⁶⁸Ga-NODAGA-exendin-4 accumulation, 12 mg/kg of unlabeled exendin-4 peptide (ChinaPeptides Co. Ltd., Shanghai, China) was injected 10 minutes before ⁶⁸Ga-NODAGA-exendin-4 injection in order to block specific binding sites 1 week after coronary ligation. The rats underwent PET/CT imaging followed by autoradiography study.