The working solution of Matrigel was prepared at a concentration of 0.5 mg/ml in PCR water, adding 100 μl to each insert and allowing to dry overnight . Once dried the inserts were rehydrated in 100 μl sterile water for 1 hour. The water was then aspirated and cells were seeded in the inserts over the top of the artificial basement membrane at a density of 30.000 cells in 200 μl Selleckchem AZD3965 per well. The plates were then incubated for 3 days at 37°C with 5% CO2. After the incubation period, the Matrigel layer together with the non-invasive cells was cleaned from the inside of the insert with a tissue paper. The cells which
had migrated through BVD-523 purchase the Matrigel and porous membrane were fixed in 4% formaldehyde (v/v) in BSS for 10 minutes before being stained in 0.5% crystal violet (w/v) in distilled water. The cells were then visualized under the microscope under X40 magnification, 5 random fields counted and duplicate inserts were set up for each test sample. In vitro Cytodex-2-bead motility assay Cells were pre-coated onto Cytodex-2 beads (GE Healthcare, Cardiff, UK) for 2 hours . The medium was aspirated and the beads were washed 2X in growth medium to remove non-adherent or
dead cells. After the second wash the beads were resuspended in 5 ml of normal growth Phosphoprotein phosphatase medium. Cell were aliquoted into a 24-well plate, 5 duplicate wells per sample (300 μl/well), and incubated overnight. Following incubation, any cells that had migrated from the Cytodex-2 beads and adhered to the base of the wells were washed gently in BSS, fixed
in 4% formaldehyde (v/v) in BSS for 10 minutes before being stained in 0.5% crystal violet (w/v) in distilled water. Five random fields per well were counted under microscope. Wound healing assay Forty thousand cells were seeded in a 24 well plate, and upon reaching confluence, the medium was changed and the monolayer was scraped with a fine gauge needle to create a wound. The plate was placed on a heated plate to keep a constant temperature of 37°C. Cells were photographed after wounding and every 15 minutes during 1 hour with a CCD camera attached to a microscope at X20 magnification . ECIS The 1600R model of the ECIS (electric cell-substrate impedence sensing) Bafilomycin A1 instrument (Applied Biophysics Inc, NJ, USA) was used for motility assay (wounding assay), wounding/cell modelling analysis in the study model. The ECIS instrument measures the resistence/impedance and capacitance of cells attached to a gold electrode. Cell modelling was carried out using the ECIS RbA modelling software, supplied by the manufacturer .The 8 W10 arrays (8 well format with 10 probes in each well) were used in the present study.