Transwell-Based Cell Migration Assay

Migration assay was performed with transwell chambers (8 µm pore size, Corning, Australia). Cells grown as spheres and monolayer were enzymatically dissociated to single cells with Trypsin-EDTA treatment. Trypan Blue dye (Thermofisher, Australia) staining was performed to determine cell viability and cell count. 1 × 10⁵ of harvested cells from sphere or monolayer culture were plated into the upper chamber in a serum-free DMEM culture medium. A total of 500 µL of DMEM culture medium with 10% FBS was added into the lower chamber as the chemoattractant. Following 24 h of incubation at 37 °C, cells were treated with 4% paraformaldehyde (Fisher Scientific, Victoria, Australia) for 15 min for fixation. Next, cells were stained with 0.1% Crystal Violet (Sigma-Aldrich, New South Wales, Australia). The cells were removed from the upper chamber. Cells migrated onto the lower chamber were photographed under an inverted Olympus DP21 microscope equipped with a digital camera. For the quantification of cell migration, the Crystal Violet staining on the transwell membrane was extracted using 5% SDS and a 570 nm wavelength was used to measure absorbance [61 (link)].