Imaging and Quantifying Cellular Dynamics

Cells grown on 12-mm No. 1.5 coverslips (Carolina Biological Supply) were fixed with 4% paraformaldehyde (Electron Microscopy Sciences), washed with PBS, and permeabilized with either 0.1% TX100 or 0.1% saponin in a 3% BSA/PBS buffer. Subsequent primary and secondary antibody incubations were carried out in the permeabilization buffer. Coverslips were mounted in ProLong Gold reagent supplemented with DAPI (Invitrogen). Images were acquired with either a Zeiss LSM 800 laser scanning confocal microscope with a 63× Plan Apo (NA = 1.4) oil immersion objective and Zeiss Efficient Navigation software or an UltraVIEW VoX spinning disk confocal microscope (PerkinElmer) that consisted of a Nikon Ti-E Eclipse inverted microscope equipped with 60× CFI PlanApo VC, NA 1.4, oil immersion objective and a CSU-X1 (Yokogawa) scan head that was driven by Volocity (PerkinElmer) software. For live cell imaging analysis of microtubules and F-actin, the cells were labeled with SiR-tubulin and SiR-actin (Cytoskeleton Inc.) as per the manufacturer’s instructions. An automated analysis pipeline was developed with Cell Profiler (McQuin et al, 2018 (link)) for the quantification of nuclear versus cytoplasmic ratios of the NLS-td-Tomato-NES reporter (Zhang et al, 2015 (link)).