CELL MIGRATION AND INVASION ASSAYS

Breast cancer cell migration and invasion assays were performed as described previously, with minor modifications (Li et al., 1998; Song et al., 2009). The migration assay was carried out in 6.5-mm diameter transwell chambers with pore size of 8.0 μm from BD Biosciences. MDA-MB-231 and BT549 cells (1 × 10⁵) in 200 μl of the serum-free DMEM medium with 0.1% BSA and 2 mM L-glutamine were placed in the upper compartment of the transwell chambers. The lower compartment was filled with 600 μl of complete medium (DMEM medium with 10% FBS and 2 mM L-glutamine). After incubation for 15 h at 37°C, cells on the lower surface of the filter were fixed in 4% paraformaldehyde and stained with crystal violet. The images were scanned and counted at 100 × magnification.

Invasion of cells through Matrigel was determined using 24-well BD invasion chambers (8.0-μm pore size with polycarbonate membrane; BD Biosciences) according to the manufacturer’s instructions. MDA-MB-231 and BT549 cells (5 × 10⁴) in 100 μl of the serum-free DMEM medium with 0.1% BSA and 2 mM L-glutamine were placed in the upper compartment of the invasion chambers, and the lower compartment was filled with 600 μl of complete medium. After incubation for 22 h at 37°C, cells on the lower surface of the filter were fixed, stained and counted at 100 × magnification.