Cells were counted using Cellometer Auto 1000 Bright Field Cell Counter (Nexcelom) and 100.000 cells were seeded on 10 µg/ml fibronectin-coated glass bottom 35 mm. Halo-CAAX was labeled using Janelia
Fluor 549 HaloTag ligand (200 -400 nM; GA1110; Promega) and endogenous SNAP-Profilin-1 were labeled using SNAP-Cell 647-SiR (500 nM -1 µM; S9102S; NEB), 30 minutes prior to imaging. Cells were imaged in phenol-red free DMEM supplemented with 20 mM HEPES pH 7.4 on a climate-controlled (maintained at 37 oC), fully motorized Nikon Ti-Eclipse inverted microscope equipped with Perfect Focus System, an Andor Diskovery illuminator coupled to a Yokogawa CSU-XI confocal spinning disk head with 100 nm pinholes, and a 60x (1.49 NA) APO TIRF objective (Nikon) with an additional 1.8x tube lens, yielding a final magnification of 108x (Andor Technology). Images were recorded at 3 Hz frame rate using a scientific CMOS camera with 6.5-µm pixel size (pco.edge).
Fluor 549 HaloTag ligand (200 -400 nM; GA1110; Promega) and endogenous SNAP-Profilin-1 were labeled using SNAP-Cell 647-SiR (500 nM -1 µM; S9102S; NEB), 30 minutes prior to imaging. Cells were imaged in phenol-red free DMEM supplemented with 20 mM HEPES pH 7.4 on a climate-controlled (maintained at 37 oC), fully motorized Nikon Ti-Eclipse inverted microscope equipped with Perfect Focus System, an Andor Diskovery illuminator coupled to a Yokogawa CSU-XI confocal spinning disk head with 100 nm pinholes, and a 60x (1.49 NA) APO TIRF objective (Nikon) with an additional 1.8x tube lens, yielding a final magnification of 108x (Andor Technology). Images were recorded at 3 Hz frame rate using a scientific CMOS camera with 6.5-µm pixel size (pco.edge).