Growth factor reduced matrigel coated 8 μm pet transwell chambers

Experiments were carried out essentially as described8 (link) using Growth Factor Reduced Matrigel-coated 8 μm PET transwell chambers (Corning). Tumour cells were seeded in duplicates onto the coated transwell filters at a density of 0.5–1 × 10⁵ cells per well in medium containing 0.8% FBS, 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 analysed by fluorescence microscopy. Five random fields at × 20 magnification were collected for each membrane. Digital images were batch imported into ImageJ, thresholded and analysed with the Analyze particles function. Experiments were repeated three times. Where indicated, cell proliferation was assayed under identical conditions by a CellTiter 96 AQueous One Solution Cell Proliferation Assay or by direct counting of cells.