Fluorescence Microscopy Protocol for Live-Cell and Immunofluorescence Imaging

Fluorescence microscopy was essentially carried out as described previously [41 (link)]. The following filter cubes were chosen to match the properties of the fluorescent reporters used in this study: F400 (excitation: 390–410 nm; dichroic mirror: 505 nm; emission: 510–550 nm), F440 (excitation: 422–432 nm; dichroic mirror: 600 nm; emission: 610 long pass), F482 (excitation: 470–495 nm; dichroic mirror: 505 nm; emission: 510–550 nm), and F562 (excitation: 545–580 nm; dichroic mirror: 600 nm; emission: 610 nm long pass). For live-cell imaging, cells were seeded and imaged in FluoroDish cell culture dishes (World Precision Instruments, Hertfordshire, UK; FD-35). To enhance the adherence of the Flp-In T-REx 293 cells to the glass surface, the dishes were precoated with polyethyleneimine (PEI) (MP Biomedicals, 195444) at 25 μg/mL in 150 mM NaCl (2 h, room temperature) [33 (link)]. For immunofluorescence microscopy, the samples were fixed and processed as described elsewhere [42 (link)]. Nanobody-based plasma membrane translocation assays were carried out as described elsewhere [39 (link)]. cellSens Dimension software (version 2.1) (Olympus Belgium) was used for image acquisition and analysis.