8 μm polycarbonate membrane filters

In vitro cell migration assays were performed in a 24-well Transwell plate with 8-μm polycarbonate membrane filters (Corning) separating the lower and upper culture chambers. RWPE-1 cells were grown to subconfluence (~75%–80%) and were incubated with LNCaP or DU145-derived exosomes (100 μg/ml) or serum-free medium for 48 hours. After detachment with trypsin, cells were washed with PBS and resuspended in serum-free medium, after which the cell suspension (1 × 10⁵ cells), supplemented with exosomes (100 μg/ml) or serum-free medium, was added to the upper chamber. Medium containing 10% FBS and exosomes (100 μg/ml) or serum-free medium was added to the bottom wells of the chamber. The cells that had not migrated were removed from the upper face of the filters using cotton swabs, and the cells that had migrated to the lower face of the filters were fixed with methanol and stained with 0.5% crystal violet solution. Images of at least 10 random fields were captured from each membrane using a ×10 objective, and the number of migratory cells was counted. All values are representative of at least two independent experiments.

Transwell chambers were used to evaluate cell migration and invasion ability, but transwell chambers for cell invasion were coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Firstly, the transfected cells (5 × 10⁴ cell/well) were suspended in serum-free medium and then seeded into the upper chamber of a 24-well transwell with 8 μm polycarbonate membrane filters (Corning). The lower chamber contained 10% fetal bovine serum (FBS) as chemoattractant. Subsequently, cells were incubated for 48 h at 37 °C, the cells adhering to the lower surface were fixed with methanol and stained with crystal violet. Then, the cells were counted under the microscope and at least selected three fields.