24 well matrigel coated invasion chambers with an 8 mm pore size

For gene-silencing experiments, BPH-1 and RWPE-1 immortalized human prostate cells, obtained from the American Type Culture Collection (ATCC) and previously checked to exclude mycoplasma contamination, were transfected with 10 nM final concentration of siRNA oligonucleotides purchased from Life Technologies (Silencer Select Pre-Designed and Validated siRNAs) using Lipofectamine RNAi Max (Life Technologies) in Opti-MEM I Reduced Serum Medium without serum (Gibco) following the manufacturer’s instructions. Two days later, their invasive potential was evaluated using 24-well Matrigel-coated invasion chambers with an 8 mm pore size (BD Biosciences). For BPH-1, 6 x 10⁵ cells were allowed to invade for 72 h using 15% fetal bovine serum as a chemoattractant, whereas for RWPE-1 cells, 8 x 10⁵ cells were seeded and allowed to invade for 48 h, using bovine pituitary extract (BPE) and human recombinant epidermal growth factor (EGF) as chemoattractants. Cells that reached the lower surface were stained with crystal violet and counted under the microscope.