Ix2 dsu confocal unit

HuH7 cells stably expressing ADRP-GFP were grown in 96-well imaging plates and stained with SiR-tubulin (1 µM; tebu-bio GmbH, Offenbach, Germany) for 45 min, according to the manufacturer’s protocol. Cells were washed, mounted and imaged on an Olympus IX-81 inverted microscope (Olympus, Tokyo, Japan) equipped with an IX2-DSU confocal unit.

CHO-K1 hIR/GLUT4-myc-GFP cells were grown and treated the same way as for objective-type TIRF imaging with the following modifications. The cells were incubated with the test substances for 10 min, and then GPMV formation was initiated. After the cells were washed three times with GPMV buffer, they were incubated in GPMV-forming buffer for 1 h. All incubation steps were performed in a humidified atmosphere (≥95%) at 37 °C and 5% CO₂. Finally, the buffer with formed GPMVs was transferred into empty wells of a 96-well imaging plate. The imaging was performed on the abovementioned Olympus IX-81 inverted microscope equipped with an IX2-DSU confocal unit. A light guide-coupled illumination system (Olympus U-HGLGPS, Tokyo, Japan) with appropriate filters (FITC) was used to image GFP fluorescence. The fluorescence signal was recorded by an Orca EM-CCD camera (Hamamatsu Photonics, Herrsching, Germany).