Migration chamber

Transfected cells incubated in 6‐well plate were cultured with trypsin enzyme digesting technique. 600 µL of RPMI1640 comprising 10% FBS was further added to the bottom chamber. Cells (3 × 10⁴) were suspended in 300 µL serum‐free RPMI1640 and seeded into the upper chamber. Then incubated at 37°C for 20 hours, the cells that migrated from upper to bottom were fixed and stained for 30 minutes with 0.01% crystal violet solution in PBS. The pictures were captured by the camera under the microscope. All experiments were accomplished in triplicate. The migration chambers and invasion chambers were purchased from Corning, NY, USA.

Transwell chambers (Corning) were used for the cell migration assay following the manufacturer's instructions. Migration chambers were purchased from Corning. These chambers were placed into a 24-well plate. A filter membrane in the upper chamber (Transwell insert) separates it from the lower chamber [22 (link)]. Cells are seeded in the upper chamber and cells migrate through the membrane to the lower chamber of the culture plate that contains chemotactic stimuli (serum is often used) [23 (link)]. Thus, cells can migrate to the serum-containing side. Briefly, A549 and H1299 cells in extracellular matrix (ECM) chambers (2 × 10⁴ cells well⁻¹) were cultured. Serum-free Dulbecco's modified Eagle medium (DMEM) (300 µl) was added to the ECM layer, which was allowed to hydrate for 1 h. Then the cells were resuspended in l-DMEM and allowed to adhere for 1 h. Migration medium containing vectors from the various groups was added to the bottom chamber. After 12 h incubation, cells in the lower chamber were fixed in 100% methanol for 30 min and then stained with 0.1% crystal violet for 20 min at room temperature. Subsequently, the cells were microscopically observed and counted in five random fields of view, and the migrated cell numbers were counted.

SW480 and HCT-8 cells (1 × 10⁵) were resuspended in 200 µl medium containing 0.1% serum and then added to the upper compartment of migration chambers (Corning, USA). The bottom chamber was filled with 600 µl cell culture medium with 20% FBS as an attractant. After coculture for 72 h, the cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. The stained cells in 8 random fields were counted at × 100 magnification.

HCT-116 and SW480 cells (1 × 10⁵) were resuspended in 200 µl medium containing 0.1% serum and then added to the upper compartment of migration chambers (Costar, Corning, NY, USA). The bottom chamber was filled with 600 µl cell culture medium with 20% FBS as an attractant. After coculture for 24 h or 48 h, the cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. The stained cells in 8 random fields were counted at ×100 magnification. Three independent measurements were conducted.

Migration and invasion assays were performed in triplicate using migration chambers (8-mm pore size, Costar) with Matrigel (BD Biosciences). RL-952 cell line was transfected for 24 h in serum-free medium before seeded into the upper chambers of transwells, while the lower chambers filled with medium containing 10% charcoal-stripped FBS. The cells were placed in incubator for several hours, fixed and stained with 0.1% crystal violet. Cells on the lower surface were photographed, and five random fields of cells were counted.