Spetramax 190

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.

RT112 cells suspended in 2% serum medium (1 × 10⁵ cells per well) were seeded in 24-well Transwell plates (pore size, 8 μm; Corning). The bottom chambers were filled with or without 0.5 × 10⁵ CSF-1-induced human macrophages, and appropriate controls or designated concentrations of compounds were added to both sides of the membrane. The cultures were maintained for further 24 h, and then the non-motile cells at the top of the filter were removed 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.