Cell Migration Assay with Inhibitor

Cell migration was performed as previously reported [49 (link),50 (link)] using a 24-well Boyden chambers (Corning, NY, USA) with inserts of polycarbonate membranes (8µm pores). MDA-MB-231 and BT-549 cells (0.5 × 10⁵/well) were re-suspended in 100 µl of serum-free medium in the presence or absence of different concentration of ψRGDechi (50 µM, 10 µM, 1µM), scrambled-peptide (50µM), and blocking anti-α_vβ₃ antibody LM609 (10 μg/mL) (Millipore, Burlington, MA, USA) and were seeded in the upper chamber. After the addition of 1% FBS or 10% FBS in the lower chamber as chemo-attractants, the trans-well were put for 24 h at 37 °C in a humidified incubator in 5% CO₂. The not migrated cells were removed with cotton swabs, whereas the cells that had migrated were visualized by staining the membrane with 0,1% crystal violet in 25% methanol. Ten random fields/filter were counted under a phase contrast microscope (Leica, Wetzlar, Germany) and images were captured by a digital camera (Canon, Tokyo, Japan) attached to the microscope. All experiments were performed at least three times and the results are expressed as the percentage of migrating cells, considering the untreated control sample as 100%.