Labtek microscope dishes

Cells were imaged in eight-well Lab-Tek microscope dishes (155411, Thermo Scientific) at 37 °C in imaging buffer containing (mM): 20 HEPES, 115 NaCl, 1.2 CaCl₂, 1.2 MgCl₂, 1.2 K₂HPO₄. For HeLa cells, the imaging buffer additionally contained 10 mM glucose. MIN6 cells (if not specified otherwise) were imaged in an imaging buffer containing a stimulatory amount of glucose (20 mM).
Imaging was performed on a dual scanner confocal microscope Olympus Fluoview 1200, with × 20 (air) and/or × 63 (oil) objectives. This microscope houses two independent, fully synchronized laser scanners for simultaneous laser stimulation and confocal observation and permits capturing of cellular responses that occur during or immediately following laser stimulation. Microscope settings were adjusted to generate images displaying background fluorescence values slightly larger than zero in order to capture the complete signal stemming from the respective fluorescent dyes or proteins. Coumarin dyes were excited with 405 nm laser and emitted light was collected between 425 and 525 nm. C1-GFP and R-GECO were excited with 488 and 559 nm lasers and emitted light was collected at 500–550 nm and 570–670 nm, respectively.

1.1B4 cells were cultured in a humidified atmosphere at 37°C and 8% CO₂. The culture medium RPMI‐1640 medium (Merck) contained 2 g/L d‐glucose, 0.3 g/L l‐glutamine, and was supplemented with 10% FBS (gradient‐grade, Thermo Fisher Scientific, catalog# 16000‐044). The medium was sterile‐filtered (Millex GV, 0.22 µm) and used within 1 week after preparation. Cells were passaged at 80% confluency. For live cell [Ca²⁺]_i imaging, 1.1B4 cells were seeded into 8‐well LabTek microscope dishes (155411, Thermo Scientific). 1.1B4 cells were transfected with a plasmid encoding the Ca²⁺‐sensitive red‐fluorescent protein R‐Geco1 using lipofectamine 2000 (11668027, Thermo Scientific) 1 day prior to imaging experiments. The transfection was performed using cells attached to LabTek microscopy dishes at 60–80% confluency. After removing the medium and washing cells with DPBS, 210 μl OptiMEM medium was added to each well. Subsequently, 1.5 μl/well of lipofectamine 2000 (1 mg/ml) and 400 ng/well plasmid DNA were added to 20 μl/well OptiMEM medium, respectively. Both solutions were combined and incubated for 10 min at room temperature. 40 μl of this transfection mix were added to each well. After 6 h of incubation at 37°C in 8% CO₂, OptiMEM was exchanged by 400 μl of RPMI‐1640 medium. Cells, showing a medium expression level of R‐Geco1, were used for live cell imaging the next day.

MIN6 cells (Miyazaki et al., 1990 (link)) were cultured in a humidified atmosphere at 37°C and 8% CO₂. The culture medium DMEM contained 4.5 g/L glucose and was supplemented with FBS (15%, gradient‐grade, Thermo Fisher Scientific, catalog# 16000‐044) and β‐mercaptoethanol (70 µM). The medium was sterile‐filtered (Millex GV, 0.22 µm) and used within 1 week after preparation. For imaging, MIN6 cells were seeded into 8‐well LabTek microscope dishes (155411 Thermo Scientific) or on 40 mm coverslips (Menzel Gläser). For in vitro assays, MIN6 cells were seeded on ∅ 60 mm or ∅ 35 mm dishes (Nunc delta surface, cat# 150288/cat# 153066; Roskilde, Denmark) to form pseudoislets within 5 days after seeding. MIN6 cells were used exclusively from passages 26 to 36.