Polycarbonate membrane

The invasion potential of the UROtsa parent cells, the As⁺³-and Cd⁺²-transformed cells, the SPARC and DEST-transfected UROtsa cells and the breast cancer cell line Hs578t (positive control) was assessed using basement membrane coated 24-well trans-well inserts. Cells were pretreated with MMC for 2 h before they were trypsinized and added to the upper chamber of the basement membrane coated 8 μm pore size polycarbonate membrane (Cell Biolabs) insert. The same concentrations of MMC were used as in the wound assay. 7.2 x10⁴ cells in 300 μL serum free media was added to the upper chamber and 500 μL of media containing 10% fetal calf serum was added to the bottom chamber of each well. The cells were allowed to invade for 24 h at 37°C, 5% CO₂: 95% air atmosphere. Cells were stained and counted in the same manner as the chemotaxis migration assay. The assay was performed in duplicate and the percentage of cells that invaded was determined.

Culture mouse germ cells were harvested and depleted of feeder cells by two rounds of preplating. 10⁵ mouse germ cells in single cell suspension were then placed in the upper chamber of a transwell insert containing a polycarbonate membrane with 5 μm pores (Cell Biolabs Inc.). The transwell insert was placed into a well of a 12-well plate containing serum-free medium with or without recombinant CXCL12/SDF-1 (R&D systems; 10 ng/mL) and AMD3100 (Sigma Aldrich, 1.25 μM) [19 (link)]. Cultures were incubated for 24 hrs following which the media containing migrated germ cells were collected and cells stained with cell stain solution (Cell Biolabs Inc.). The absorbance of invading germ cells was quantified at OD at 562 nm. cell stain solution in blank wells containing only medium was used as the reference solution. The higher the number of invading germ cells in the bottom well is, the greater the absorbance quantified is.