BD Matrigel Invasion Assay
BD Matrigel™ Matrix is a solubilized basement membrane matrix extracted from the EHS mouse tumor, a tumor rich in basement membrane proteins, Matrigel Matrix has been extensively used as the in vitro barrier to study metastatic potential of tumor cells.
Growth Factor Reduced Matrigel™ Matrix ‘s gelling is reversible. It will gel rapidly at 22°C to 35°C. Gelled Matrigel may be re-liquified if placed at 4°C on ice for 24-48 hours.
Matrigel ( Growth Factor Reduced BD Matrigel™ Matrix (cat#: 356230 (5ml) or 354230 (10ml)) @ -20oC 24-transwell (Coster) Fibronectin Diff-Quick staining solution (IMEB, order through Fischer Scientific) Cotton swab Pipettes
Thaw Matrigel at 4°C overnight on ice . Chill pipettes, plates and tips at -20°C . Dilute Matrigel (5mg/ml) in serum-free cold DMEM with pre-cooled pipetes. 100 ul (h=100ul/200mm 2=0.5mm) of the diluted matrigel per well may be gently pipetted using a pre-cooled pipette to ensure homogenecity. Incubate the transwell at 37 oC for 30min to 5 h for gelling. Harvest cells from tissue culture flasks by Trypsin/EDTA. Wash the cells 3 times with DMEM containing 1 % FBS. Resuspend the cells in media containing 1% FBS at a density of 10 6cells/ml. A series of different density of cells may be used if necessary, especially the first time to do the invasion. Gently wash gelled matrigel with warmed serum free-culture media. Put 100 ul of the cell suspension onto the matrigel. Lower chamber of the transwell is filled with 600 ul (3mm) of culture media containing 5 ug/ml fibronectin, as an adhesive substrate. Incubate at 37 oC for 20 to 24 h or up to 40 h depending on cell species. Remove transwells from 24-well plates and stain with Diff-Quick solution. Scrape off noninvaded cells on the top of the transwell with a cotton swab. Count invaded cells on the transwell under a light microscope.