Mouse MIN6 Cell Culture and Microscopy

Mouse MIN6 cells (a kind gift from the Miyazaki laboratory, Osaka University) were cultured between passage number 25 and 35. β-Mercaptoethanol was added freshly to the medium consisting of DMEM (Lifetechnologies), 1% penicillin/streptomycin (Gibco), 15% Fetal Bovine Serum (FBS, Gibco). Cells were maintained under 8% CO₂ at 37°C and subcultured as previously described(Ishihara et al., 1993 (link), Nakashima et al., 2009 (link), Miyazaki et al., 1990 (link), Cheng et al., 2012 (link)). For microscopy experiments, cells were seeded on slides within eight-well Lab-Tek Chambered Coverglass systems (Thermo Scientific) at 60-80% confluency. MIN6 cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 24 h the transfection medium was replaced by culture medium followed by another 24 h incubation period. For the experiments indicated, medium was exchanged prior to imaging and cells were allowed to adapt 30 min for to imaging buffer IB (115 mM NaCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 1.2 mM K₂HPO₄, 0.2% glucose and 20 mM HEPES, pH 7.4). Tolbutamide (Sigma) was dissolved in DMSO and applied for 30 min at 100 μM (100 mM stock in DMSO).