Investigating Cell Invasion and Migration Mechanisms

Cell invasion assays were performed using transwell migration chambers, as described previously^{13 (link)}. Briefly, 4175 and 4T1 cells were pretreated with 1 µM RAGE inhibitors (FPS-ZM1 or TTP488) or 0.1% DMSO for 1 h in culture media. 4175 cells (1.5 × 10⁴ cells) and 4T1 cells (6.5 × 10⁴ cells) were seeded in the upper chamber of 8-μm porous transwell inserts (cat# 662638 ThinCerts; Greiner bio-one) coated with 12.5 μg of Growth Factor Reduced Matrigel matrix (cat# 354230, Corning), in serum-free DMEM or RPMI, and incubated in 24-well plates with 1% FBS as a chemoattractant for 24 h. RAGE inhibitors or DMSO control were added to the appropriate complete media in both the upper and lower chambers. Following incubation for 16 h, cells were fixed with methanol for 10 min and stained with 0.1% crystal violet in dH₂O. Non-invaded cells and excess stain were rinsed with water and removed from the inner surface of the insert with a cotton swab. To quantify the invaded cells, the insert membranes were imaged at 2x magnification (SteREO Discovery microscope, Zeiss), and the invaded area % was quantified with ImageJ 1.34n software (National Institutes of Health, USA). To assess cell migration, the same transwell assay method was used without Matrigel coating of the transwell inserts, as we have previously reported^{42 (link),70 (link)}.