Transwell permeable support inserts

For invasion assays, 50 μL of a 1:5 dilution of Matrigel in serum free RPMI was plated on Costar Transwell Permeable Support inserts with 8 μm pore size and allowed to solidify at 37°C for 2 hours. Matrigel was re-hydrated with 50 μL serum free media for an additional 30 minutes at 37°C. Un-coated Transwell Inserts were used for motility assays. 600 μL complete media containing vehicle control or drug was placed on the underside of the insert. 1×10⁴ cells diluted in a total volume of 100 μL serum free media plus or minus drug treatments were seeded on top of the insert. Cells were allowed to migrate or invade for 48 hours then cells were fixed with 4% PFA for 20 minutes and stained with 0.1% crystal violet for 20 minutes. Cells and Matrigel remaining on the top of the Transwell insert were removed with a cotton swab and cells on the underside of the Transwell insert were imaged using an Olympus CKX41 microscope with DPManager software. Representative 10X images are shown. Transwell invasion and motility assays were quantified by counting the number of cells on the underside of the insert per 10X field from three independent experiments.