Matrigel-based Transwell Invasion Assay

Experiments were carried out essentially as described (16 (link)) using Growth Factor Reduced Matrigel-coated 8 μm PET transwell chambers (Corning). Tumor cells were seeded in duplicates onto the coated transwell filters at a density of 1×10⁵ cells/well in medium containing 0.1% BSA and conditioned media from NIH3T3 was placed in the lower chamber as chemoattractant. Cells were allowed to invade for 16–24 h, non-invading cells were scraped off the top side of the membranes and the invasive cells on the transwell insert were fixed in methanol. Membranes were mounted in medium containing DAPI (Vector Labs) and analyzed by fluorescence microscopy. Five random fields at 10X magnification were collected for each membrane. Digital images were batch imported into ImageJ Fiji, thresholded and analyzed with the Analyze particles function. Experiments were repeated three times.