Fibronectin coated polycarbonate membrane

A total of 1 × 10⁵ (for C666-1, SUNE-1) or 4 × 10⁴ (for CNE-1 and CNE-2) cells in 200 μl of serum-free RPIM 1640 were transferred to the superstratum chamber inserts with 8 μm pore size fibronectin-coated polycarbonate membrane (Corning, Corning, NY, USA) and 750 μl of RPIM 1640 containing 20% FBS was added into the chamber below. CNE-1, CNE-2, SUNE-1 cells were incubated for 18 h, respectively, while C666-1 cells were incubated for 24 h. Then the upper chamber soaks in 4% PFA Fix Solution (JingXin, Guangzhou, China) for 15 min with removing the cells on the higher surface of the membrane, after the sample was dried stained with crystal violet for 10 min, and then the number of cells on the membrane was counted by inverted microscope (×100 magnification). Cell invasion assays were performed as the migration assay, except the upper membrane was coated with Matrigel before the step seeds cell 3 h (BD Biosciences, Franklin Lakes, NJ, USA).

A total of 8 × 10⁴ (for KYSE140) or 4× 10⁴ (for KYSE510) cells in 200 μl of serum-free RPIM 1640 were transferred to the superstratum chamber inserts with 8 μm pore size fibronectin-coated polycarbonate membrane (Corning, Corning, NY, USA) and 750 μl of RPIM 1640 containing 20% FBS was added into the chamber below. KYSE140 cells were incubated for 36 h, respectively, while KYSE510 cells were incubated for 24 h, and then the upper chamber was soaked in 4% PFA Fix Solution (JingXin, Guangzhou, China) for 20 min with removing the cells on the higher surface of the membrane, after the sample was dried stained with crystal violet for 15 min, and then the number of cells on the membrane was counted by inverted microscope (×100 magnification). All experiments were repeated three times unless indicated otherwise. Cell invasion assays were performed as the migration assay, except that the upper membrane was coated with Matrigel before the step seeds cell 4 h (BD Biosciences, Franklin Lakes, NJ, USA).