Cells were seeded on a gelatin-coated 22 × 22–mm cover glass (Corning; 2850-22) overnight and moved to fresh cell culture medium 30 min before being mounted on a 25 × 75–mm slide (Thermo Fisher Scientific; 3050). Imaging was conducted with a 60×, 1.49-NA Apo TIRF objective (Nikon) mounted on a Ti-Eclipse inverted microscope. Perfect focus was used during time-lapse imaging. TIRF penetration depth was ∼80 nm. Videos were acquired for 7.5 min at the rate of 1 frame/s for all live-cell imaging. For dual-color imaging, two sequential images of primary and secondary channel were taken within 1 s, and the video length was the same as single-channel imaging. Published cmeAnalysis software was used for CCP detection, tracking, and quantification (Aguet et al., 2013 (link); Jaqaman et al., 2008 (link); Loerke et al., 2011 (link)). More than 10 cells were imaged per condition, and the number of total analyzed tracks is indicated in the figure legends.
Free full text: Click here
