24 well transwell invasion assay plate

About 5×10⁴ cells in 200 µl of α-MEM (Gibco, Life Technologies, Monza, Italy) were plated in the Matrigel-coated upper chambers of the 24-well Transwell invasion assay plate (Corning, NY 14831 USA). Plates were incubated at 37°C for 48 h. Cells in the lower chamber (including those attached to the lower surface of the membrane) were fixed in 4% paraformaldehyde (VWR, Milan Italy), stained with DAPI (Lonza, Basel, Switzerland), and counted with fluorescence microscope (Metamorph – Axiovert microscope).

Boyden chamber and 3D Matrigel invasion assays were performed as previously described53 (link). Briefly, cells (5 × 104 in 200 μl of α-MEM) were plated in the Matrigel-coated upper chambers of the 24-well Transwell invasion assay plate (Corning). Each condition was represented in triplicate. Plates were incubated at 37 °C for 24 h for HCC1806 cells. Cells in the lower chamber (including those attached to the under surface of the membrane) were trypsinized and counted using a Casy 1 counter (Sharfe System). Mean and s.e.m. of independent experiments were calculated. Statistical analysis was performed using a two-tailed Student's t-test to determine the statistical significance of the differences observed. A P value below 0.05 was considered significant.