Millicell cell culture inserts 24 well plates

Cell migration and invasion assays were performed using Millicell Cell Culture Inserts (24-well plates; 8 µm pore size; Merck KGaA, Darmstadt, Germany). Stably transduced cells were used for these assays. For the migration assay, 5×10⁴ HuCCT-1 cells or 5×10⁴ RBE cells in serum-free medium were seeded on the upper chambers. For the invasion assays, the membranes of the upper chambers were coated with 4 µl Matrigel (BD Biosciences) in 36 µl RPMI-1640 medium for 2h in a humidified incubator. The cells were then seeded in the coated upper chambers. RPMI-1640 medium containing 10% FBS was added into the lower chambers. HuCCT-1 cells and RBE cells were incubated for 24 or 48h for the migration assays, and 48 or 72h for the invasion assays, respectively. Then, the cells on the lower membranes were stained using a Wright-Giemsa Stain kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and observed at ×100 magnification. Five fields were randomly chosen and cells were counted upon observation under a light microscope, the number of cells of average field was calculated finally.

Cell migration and invasion assays were performed using Millicell Cell Culture Inserts (24-well plates; 8 µm pore size; Merck KGaA, Darmstadt, Germany). Stably transduced cells were used for these assays. For the migration assay, 3×10⁴ CCLP-1 cells or 5×10⁴ HCCC-9810 cells in serum-free medium were seeded on the upper chambers. For the invasion assays, the membranes of the upper chambers were coated with 8 µl Matrigel (BD Biosciences) in 32 µl RPMI-1640 medium for 3 h in a humidified incubator. The cells were then seeded in the coated upper chambers. RPMI-1640 medium containing 10% FBS was added into the lower chambers. CCLP-1 cells and HCCC-9810 cell were incubated for 24 or 48 h for the migration assays, and 48 or 72 h for the invasion assays, respectively. Then, the cells on the lower membranes were stained using a Wright-Giemsa Stain kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and observed at ×100 magnification. Five fields were randomly chose and cells were counted upon observation under a light microscope, the number of cell of average per field was calculated finally.