Matrigel coated 24 well transwell chambers

Breast cancer cell invasion was assessed using Matrigel™-coated 24-well Transwell^® chambers (Corning Inc., Corning, NY, USA). Polycarbonate filters with a pore size of 8 μm, which separated the upper and lower compartments, were coated with 50 μg reconstituted basement membrane. At 48 h after transfection, serum-free RPMI-1640 containing 2×10⁵ cells in a volume of 300 μl was added to the upper compartment and the RPMI-1640 in the lower compartment was supplemented with 15% fetal calf serum. Cells in upper compartment were allowed to migrate to the lower compartment for 24 h at 37°C, after which the non-invasive cells on the upper surface of the membrane were removed using cotton swabs. The invasive cells attached to the lower membrane surface were fixed using 4% paraformaldehyde and stained with hematoxylin and eosin (H&E). The number of invasive cells was counted using a microscope (Olympus, Tokyo, Japan; magnification, ×200).

Matrigel-coated 24-well Transwell chambers (Corning Incorporated, Corning, NY, USA) and non-coated Transwell chambers (#353097; BD Biosciences, USA) were separately used for cell invasion assay and cell migration assay. Twenty-four hours after transfection, the cells were plated into the upper compartment containing serum-free DMEM at a density of 2 × 10⁴ cells per well. The lower chamber was added with DMEM containing 20% FBS. Forty-eight hours later, the cells in the upper chamber were removed with cotton swabs, while the cells on the lower surface were fixed with 4% paraform-aldehyde for 10 min and stained with 0.1% crystal violet for 15 min. The results were obtained using an inverted microscope (Olympus, Japan).

Cells were plated in the upper of matrigel-coated 24-well transwell chambers (Corning, New York, USA) and cultured with the serum-free medium while the lower chamber was with medium containing 10% FBS. After incubating with CAPE for 24h or transfected with siRNA of MD2 for 24h or FGFR1 plasmid for 12h, noninvasive cells on the upper surface were removed with cotton-tipped swabs while invasive cells on the lower surface of the membrane were stained with crystal violet. Images were captured with light microscopy (Nikon, Tokyo, Japan).