Visualizing Vimentin Dynamics in Cells

A total of 150,000 cells were seeded on 25 mm high-precision No. 1.5 borosilicate coverslips (Marienfeld) in 6 well plates (ThermoFisher Scientific) 1 day before transfection in DMEM w/o phenol red (see cell culture). Each chamber was transfected with 2 µg of the plasmid pMD-Vim-Dreiklang^{17 (link)} using Lipofectamine 3000 (ThermoFisher Scientific) according to manufacturer’s instructions and imaged about 24 h post transfection. Before imaging, the coverslip was incubated with 600 nM Mitotracker Deep Red FM in DMEM w/o phenol red for ~30 min at 37 °C and 5% CO₂, followed by 10 min incubation of ~10 µg ml⁻¹ WGA-AbberiorFlip565 in Hanks balanced salt solution w/MgCl₂ and CaCl₂ (HBSS, Gibco). After 1× washing in HBSS the samples were imaged in HBSS at RT for up to 30 min. For imaging, we preswitch Dreiklang using 488 nm light for 10 s before starting data acquisition.