HCT116 and HT29 cell migration was analyzed using the in vitro wound healing assay. Cells were grown to confluence in 48-well plates and changed to serum-free medium for additional 24 h. Cell monolayers were scraped with a micropipette tip and treated with 10 uM IWR-1. The wound area was photographed under phase-contrast microscopy before and 24 h after the treatment and the percentage of wound closure was determined as: [(initial area - final area)/initial area] × 100 [64 (link)].
Invasion assays were conducted using the CytoSelect 24-Well Cell Invasion Assay Kit (Cell Biolabs, San Diego, CA, USA). Briefly, 300 μL of HCT116 and HT29 cells (1 × 105cells/ml) in serum-free medium was plated into the CytoSelect basement membrane chamber, respectively, and 500 μL of 10% FBS-containing RPMI was added to the lower well of the invasion plate; both upper and lower chambers contained the 10 uM IWR-1. The chambers were then incubated for 48 h at 37°C in a 5% CO2 atmosphere, non-migratory cells were removed, and migrated cells were stained, dissociated from the membrane, and their absorbance was measured at 560 nm using the microplate reader (model 680; Bio-Rad, Hercules, CA, USA) [65 (link)].
Invasion assays were conducted using the CytoSelect 24-Well Cell Invasion Assay Kit (Cell Biolabs, San Diego, CA, USA). Briefly, 300 μL of HCT116 and HT29 cells (1 × 105cells/ml) in serum-free medium was plated into the CytoSelect basement membrane chamber, respectively, and 500 μL of 10% FBS-containing RPMI was added to the lower well of the invasion plate; both upper and lower chambers contained the 10 uM IWR-1. The chambers were then incubated for 48 h at 37°C in a 5% CO2 atmosphere, non-migratory cells were removed, and migrated cells were stained, dissociated from the membrane, and their absorbance was measured at 560 nm using the microplate reader (model 680; Bio-Rad, Hercules, CA, USA) [65 (link)].
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