Quantifying Cell Invasion Potential

In vitro invasion assays were performed as previously described (Bravo-Cordero et al., 2011 (link)). In brief, 5 × 10⁵ cells were suspended in low (0.25%)-serum culture medium and plated in duplicate in the top well of growth factor–reduced Matrigel-coated invasion chambers (8-µm pore size; BD BioCoat; 354483; BD). Normal serum-containing media (5% serum) were placed in the lower chamber, and cells were allowed to invade for 24 h at 37°C. (For MDA–MB-231 cells, the low-serum culture media contained 0.5% serum, whereas normal growth media contained 10% serum.) The assay was fixed with 3.7% PFA for 20 min and then stained with DAPI or NucBlue Live Cell Stain ReadyProbes reagent (R37605; Molecular Probes) to visualize nuclei. Four to six random fields were acquired at 20× magnification on an SP5 confocal microscope (Leica Microsystems). The number of invaded cells was counted manually with ImageJ software and normalized to the total number of cells present in the insert. Data are means of three independent experiments for each condition.