Quantifying PC3 Cell Invasion

After transfection, the invasive behavior of PC3 cells was measured using the BD BioCoat Matrigel Invasion inserts [29 (link)]. Cell culture inserts (VWR International, Bridgeport, NJ) were coated with 50 μl of 1:4 Matrigel/Medium dilutions (BD Sciences, San Jose, CA) and allowed to solidify at 37°C for 1 hr. Cells were resuspended (5.0 × 10⁴ cells/ml) in MEM and 0.1% FBS and 500 μl of cell suspension was added to each insert. Chemoattractant solutions were made as described above with TGF-β1 and EGF into MEM supplemented with 0.1% FBS. Matrigel and non-invading cells were removed by scrubbing. Invading cells in the membrane were fixed in 3.7% paraformaldehyde and stained with DAPI. Pictures were taken from five different fields for average number of invading cells to be determined. The results were expressed as an invasive index defined as: the average number of cells per field for test substance/the average number of cells per field for the medium control. The experiments were conducted at least three times using independent cell preparations.