2.8. Primary Islet Cell Culture and Transfection

Pancreatic islets were isolated from 10- to 11-week-old C57BL/6J mice by retrograde injection of collagenase (Sigma) into the pancreatic duct according to the standard procedure, as described previously (Imai et al., 2008, Gotoh et al., 1985). The freshly isolated islets were dissociated into dispersed islet cells by trypsinization and distributed into 96-well plates (40 islets per well) and maintained in RPMI1640 medium containing 10% fetal bovine serum, penicillin-streptomycin and gentamicin at 37 °C and 5% CO₂ for 2 days. Then, islet cells were co-transfected with 10 pmoles of miR-106b mimics and 10 pmoles of miR-222 mimics (Ambion), or transfected with 20 pmoles of non-targeting control (Ambion) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. Three days after transfection, the cells were collected and analyzed for Ki-67 mRNA expression.