Cellsens dimension software version 2.1

Fluorescence microscopy was carried out as described previously [15 (link),60 (link)] using the following filter cubes: F400 (excitation: 390–410 nm; dichroic mirror: 505 nm; emission: 510–550 nm), F425 (excitation: 422–432 nm; dichroic mirror: 600 nm; emission: 610 long pass), F480 (excitation: 470–495 nm; dichroic mirror: 505 nm; emission: 510–550 nm), and F565 (excitation: 545–580 nm; dichroic mirror: 600 nm; emission: 610 nm long pass). The cells were seeded and imaged in FluoroDish cell culture dishes (World Precision Instruments, Hertfordshire, UK; FD-35) precoated with 25 μg/mL polyethyleneimine (MP Biomedicals, 195444) [46 (link)], and cellSens Dimension software (version 2.1) (Olympus Belgium) was used for image acquisition and analysis.

Following the migration assay of SaOS-2, SaOS-LM2, and HT-1080, transwell supports were washed twice with Dulbeco’s phosphate-buffered saline (Gibco, Cat.# 14190144) and cells that did not migrate were removed from the upper chamber using a moistened cotton swab. Migrated cells that adhered to the lower surface of the support were stained with crystal violet (Fisher Scientific, Cat.# C58125) for 10 min. Transwell supports were washed with Dulbeco’s phosphate-buffered saline three times and air-dried. Once dry, bright field images were taken using Olympus cellSens Dimension software (Version 2.1) on an Olympus SZX16-ILLT microscope (Olympus, Tokyo, Japan) with 5× objective lens and 2.9× zoom. The crystal violet bound to migrated cells was eluted from the supports by pipetting 400 µl of 33% acetic acid (Fisher Scientific, Cat.# A38500) into each upper chamber and shaking the plates for 10 min. Half of the eluent for each sample, 200 µl, was transferred to a 96-well clear microplate (Corning, Cat.# 3595) and the absorbance at 590 nm was determined using the Tecan Infinite M200 plate reader (Tecan Group Ltd., Männedorf, Switzerland) and Tecan i-control software (Version 3.91.0). Standard curves were generated for each cell line used in the migration assay and were used to calculate the cell concentration from the experimental absorbance measurements.

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.