Inverted spinning disk confocal microscope

Glass coverslips (TH. Geyer, 25 mm diameter, No. 1.5H) were coated with poly-d-lysine solution (Sigma-Aldrich, #P6407) at a concentration of 0.1 mg ml⁻¹ for 5 min at room temperature and washed three times with PBS. Cells were seeded on poly-d-lysine-coated coverslips and live-cell microscopy was performed 12–16 h after seeding. Live-cell imaging of AP2-eGFP was performed with an inverted spinning-disk confocal microscope (PerkinElmer), with a 60× (1.42 numerical aperture, Apo TIRF, Nikon) or 100× (1.4 numerical aperture, Plan Apo VC, Nikon) oil immersion objective and a CMOS camera (Hamamatsu Ocra Flash 4). An environment control chamber was attached to the microscope to keep 37 °C and 5% CO₂. The 10-min-long movies of representative cells were taken with one frame every 3 s. Live-cell imaging of AP2-eGFP together with CLCa-tdtomato was performed with an inverted Ti microscope (Nikon) with objective TIRF illumination, with a 60× (1.49 numerical aperture, Apo TIRF, Nikon) oil immersion objective and EMCCD camera (Andor iXon Ultra DU-897U). An on-stage incubation chamber was used to keep 37 °C and 5% CO₂. The 10-min-long movies of representative cells were taken with one frame every 3 s.

U2OS wild-type or stably expressing mCherry-Gal3 were transfected with an eGFP-Rab7 (Addgene) expressing plasmid 16 h prior to imaging. MRV labeled with Alexa647 was added to cells and then imaging was started. Live-cell imaging was performed with an inverted spinning-disk confocal microscope (PerkinElmer) using oil immersion objectives (60x, 1.49 NA, Apo TIRF, Nikon or 100x, 1.4 NA, Plan Apo VC, Nikon) and a CMOS camera (Orca Flash 4, Hamamatsu). Cells, objectives and microscope stage were kept at 37°C and 5% CO₂ through the presence of an environment-control chamber. Cells were imaged in 0.5 μM stacks 5min apart for 180 min.

The 1 × 10⁴ tfRFP-B16 cells were cultured overnight in a chambered coverslip with 8 wells (ibidi, Germany). Then, 5 × 10⁵ bone marrow cells from ROSA^mT/mG mice were added with 50 μg anti-tfRFP IgG alone, or 50 μg anti-tfRFP IgG in combination with 50 μL normal mouse serum. After 4 hours, the cultured cells were imaged with a spinning disk inverted confocal microscope (PerkinElmer) with a dry 20×/0.75 NA objective (Olympus).