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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.
Diff-Quick staining solution (IMEB, order through Fischer Scientific)
Cotton swab
Pipettes
Procedure
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 37oC 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 106cells/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 37oC 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.