Biocoat matrigel invasion inserts

The invasive properties of DU145 cells were measured using the BD BioCoat Matrigel Invasion inserts. Inserts were coated with 50μl of a 1:4 Matrigel/medium dilution and allowed to solidify at 37 °C for 48 hours. Cells were resuspended (3 × 10⁴ cells/ml) in MEM with 0.1% FBS and 500μl of cell suspension were added to each insert. Cells were treated with TGF-β1 and TGF-β3 (5ng/ml), or EGF (10ng/ml) and were allowed to invade through the porous membrane coated with Matrigel for 48 hours. Matrigel and non-invading cells were removed via cotton swabs. Invading cells on the membrane were fixed in 3.7% paraformaldehyde and stained using DAPI (Roche Diagnostics, Indiana, IN). Images were taken in ten different fields for sum of invading cells. The results were expressed as invasion index defined as: the sum of ten random fields for test substance/the sum of ten random fields for the medium control.

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.

5 × 10⁴ cells were resuspended in serum-free and glucose-free DMEM and seeded into the interior of BD BioCoat Matrigel Invasion inserts (n=3/group). Once the cells were seeded, DMEM supplemented with 20% FBS was placed into the lower well as the chemoattractant. After 22 h incubation, non-invasive cells were removed from the upper surface, and the remaining cells were stained with crystal violet or the Hemacolor stain set (EMD Millipore) and then quantified.