Matrigel coated membrane inserts

Cells (2 × 10⁴ cells/0.5 ml/well) were plated onto control membrane inserts with 8-μm pores or onto Matrigel-coated membrane inserts (BD Biosciences, San Diego, CA, USA) that were placed in 24-well chambers filled with 0.6 ml of growth medium. Twenty-four hours after plating, cells that remained on the upper surface of the membrane were removed using cotton-tipped swabs, and cells that had migrated to or invaded the lower surface of the membrane were fixed with methanol, stained with 0.5% crystal violet and counted under the microscope. The migration percentage was calculated as follows: Migration (%) = (Mean number of cells invading through uncoated insert × 100)/Mean number of cells seeded onto the regulator culture surface. The invasion percentage was calculated as follows: Invasion (%) = (Mean number of cells invading through Matrigel insert membrane × 100)/Mean number of cells migrating through control insert membrane.

For the migration assay, NCI-H441 cells suspended in serum-free medium (1.5×10⁵ cells per well) were seeded in 24-well Transwell plates (pore size, 8 μm; Corning). The bottom chambers were filled with serum-free medium supplemented with HGF (100 ng/mL), and 0.8, 4, 20 and 100 nM of Simm530 was added to both sides of the membrane. The cultures were maintained for 24 h, followed by the removal of non-motile cells at the top of the filter using a cotton swab. The migrating cells were fixed in paraformaldehyde (4%) and stained with crystal violet (0.1%) for 15 min at room temperature. The dye that was taken up by the cells bound to the membrane was released by the addition of 100 μL 10% acetic acid, and the absorbance of the resulting solution was measured at 595 nm using a multiwell spectrophotometer (SpetraMAX 190, from Molecular Devices, Palo Alto, CA, USA). The assay was performed in triplicate. Images were obtained using an Olympus BX51 microscope.
For the invasion assay, NCI-H441 cells were cultured in the top chambers containing Matrigel-coated membrane inserts (Matrigel, BD). The ensuing procedure was identical to the migration assay. The assay was performed in triplicate. Images were obtained using an Olympus BX51 microscope.

Briefly, 3 × 10⁵ RCC cells were seeded on the upper surface of Matrigel-coated membrane inserts (BD Biosciences, San Jose, USA) in serum-free medium, and the lower chamber contained medium with exosomes from different sources. After 24 h of incubation, cells that had migrated from the upper part of the filter to the lower part were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet for 30 min. The invasive cells were counted and photographed in three random views.