Matrigel coated transwell membranes

The ability of cells to invade was assessed using Matrigel-coated Transwell membranes (BD Biosciences). After 30 min incubation at 37°C, the Matrigel solidified and served as an extracellular matrix for tumor cell invasion analysis. The upper chamber contained ~5×10⁴ cells in 200 µl DMEM without serum. The lower chamber contained DMEM with 10% FBS. The cells were then incubated for 48 h at 37°C in an atmosphere containing 5% CO₂. Cell invasion to the underside of the Matrigel-coated membrane was subsequently fixed with 4% paraformaldehyde for 10 min at room temperature, stained with 0.5% crystal violet for 10 min at room temperature followed by imaging and counting under an inverted microscope at ×200 magnification (Olympus Corporation). The results are expressed as the average number of invasive cells per field.

Transwell invasion assays were performed using Matrigel-coated Transwell membranes (BD Biosciences, San Jose, CA, USA). The Matrigel solidified after a 30-min incubation at 37°C and post-transfection, the cells (10⁵) in 100 µl of serum-free RPMI-1640 medium were added to the upper compartment of the chamber. As a chemoattractant, 20% FBS in RPMI-1640 medium was added to the lower chambers of the wells. The cells were then incubated at 37°C in 5% CO₂ for 48 h, and the lower cells were stained with crystal violet (Zhongshan Bio Corporation) for 5 min at room temperature. The average number of cells per field was calculated by counting cells in 6 random fields per well under the microscope at ×200 magnification (Olympus Corporation).