Ocra flash 4

Confocal live fluorescence cell imaging was performed with an inverted spinning disc confocal microscope (Nikon Instruments, PerkinElmer) equipped with 60× (1.42 numerical aperture, Apo TIRF, Nikon) oil immersion objective and a CMOS camera (Hamamatsu Ocra Flash 4) or an EMCCD camera (Hamamatsu C9100-23B). The biological samples were kept in an environment control chamber at 37 °C with 5% CO2.

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.