Ts100 100 f inverted fluorescence microscope

MCF-7 or BT-549 cells were plated on glass slides (1 × 10⁴ cells/well) and incubated for approximately 24 h at 37 °C. After treating the cells with formaldehyde and Triton-X, the cells were incubated with primary antibodies directed against E-Cad and Vimentin (dilution 1:250, Cell Signaling Technology, Inc., USA) at 4 °C overnight with gentle shaking. The following day, the cells were incubated with secondary antibody (Supplementary Table S5) for 1 h at room temperature away from light sources. Finally, the samples were stained with 4',6-diamidino-2-phenylindole (DAPI) and were observed with a TS100/100-F inverted fluorescence microscope (Nikon, Corporation, Tokyo, Japan).

Cell migration was determined by using 24-well Transwell Chambers with an 8-μm pore size and a track-etched membrane (Corning, Inc., New York, CA, USA). A chamber insert was placed into each well of a 24-well dish containing 600 μl of DMEM supplemented with 20% FBS in the lower chamber. Approximately 5×10⁴ cells were suspended in 200 μl of serum-free DMEM and seeded into the upper chamber. The cells were incubated at 37 °C with 5% CO₂ for 24 h. Then, the cells on the lower chamber membrane were fixed with 100% methanol for 30 min at room temperature and stained with 0.1% crystal violet for 20 min at room temperature. Subsequently, the nonmigrated cells on the upper side of the membranes were removed, and the migrated cells on the underside of the membranes were observed with a TS100/100-F inverted fluorescence microscope (Nikon, Corporation, Tokyo, Japan) using five randomized fields. A cell invasion assay was also conducted in the same manner but with Matrigel™ Invasion Chamber 24-well plates with 8.0-μm pores (BD Biosciences, San Jose, USA) and an incubation time of 48 h.