Transwell with 8.0 μm pore polyester membrane insert

In order to test the ability of cells migration, 6.5mm Transwell^® with 8.0 μm Pore Polyester Membrane Insert (Product #3464, Corning, Inc., Corning, NY) was used. In order to test the invasion ability of cells crossing through matrigel-coated filter, 6.5 mm Transwell^® with 8.0 μm Pore Polycarbonate Membrane Insert coated with Cultrex^® Basement Membrane Extract (BME) (Product#3432-001-01, Trevigen Inc.) were used. The procedure was performed as described previously [49 (link)]. Cells were starved for 18 h using no-serum medium and then 1 × 10⁵ cells per well were seeded in insert with 200 μl no-serum medium with or without DIM, and 800 μl of growth medium containing 5% FBS was added in bottom wells. Following a culture of 16 h, non-migrating cells were removed from the upper surface by wiping with a cotton swab. The membrane was fixed with 4% formaldehyde for 15 min at room temperature. Cells were stained with Giemsa for 25 min, and their numbers in 5 fields of each membrane were counted under an inverted microscope. The procedure of PF-562271 was the same as that of DIM, the no-serum medium containing different concentration of PF-562271 was loaded in inserts. For the co-treatment of vitronectin and DIM, the same procedures were used except that the bottom wells were loaded with no-serum medium containing 5 ng/ml vitronectin or 5 ng/ml BSA.

Cell migration assay was performed using modified Boyden chambers as described by Chen [31 (link)]. Briefly, 1 × 10⁵ cells were plated at the upper compartment of a Transwell® with 8.0 μm pore polyester membrane insert (Corning) and medium with 10% FBS was placed at the inferior compartment to act as a chemoattractive solution. Following an incubation period (3 hours for tumor pericytes and 12 hours for normal tissue pericytes), the cells that migrated through the membrane were fixed and stained with 1% of toluidine blue and 1% borax solution. Stained cells were lysed with 1% SDS, and optical density was measured at 626 nm.