8 μm pore transwell chambers in 24 well plates

Transwell assays were performed using 8-μm pore transwell chambers in 24-well plates (Corning Costar, Cambridge, MA, USA). The upper chambers were seeded with 1 × 10⁵ Hep3B cells in 100 uL of the serum-free DMEM/F12 medium. These Hep3B cells had been previously transfected with either the control pMXs-EGFP vector or the pMXs-OCT4 plasmid. The lower chambers were filled with 800 uL of the DMEM/F12 medium containing 10% FBS. Subsequently, the cells were incubated at 37°C in a 5% CO₂ humidified atmosphere for 24 h. After swabbing the upper chambers to remove cells that did not migrate, the cells that migrated to the lower chambers were fixed with 3.7% paraformaldehyde in PBS and stained using hematoxylin. The migrated cells were counted under a light microscope in five predetermined fields. The assays were performed in triplicate, and the results are expressed as the percentage of the mean of three wells containing cells transformed with the control vectors.

Transwell migration assays were performed using 8-μm-pore transwell chambers in 24-well plates (Corning Costar, Cambridge, MA, USA). After overnight starvation in serum-free RPMI-1640, the cell suspension (4 × 10⁵ cells per ml, 100 μl) was added to the upper chamber. The lower chamber was filled with 800 μl of RPMI-1640 medium containing 10% fetal bovine serum. Subsequently, the cells were cultured for another 12 h. After swabbing the non-migrated cells from the upper chambers, the cells that had migrated to the lower chambers were fixed with 4% paraformaldehyde in PBS and stained with Giemsa. Finally, the cells that had migrated to the lower chambers were observed under a light microscope (× 200). The cell invasion assay procedure was similar to that used for cell migration, except that the transwell membranes were pre-coated with 50 μg μl⁻¹ Matrigel (BD Biosciences) and the cells were incubated for 48 h. Cells that had migrated to the lower chambers were quantified as described for the cell migration assay.

8-μm-pore transwell chambers in 24-well plates (Corning Costar, Corning, NY, USA) were used. Chambers coated with Matrigel (BD, San Diego, CA, USA) were for cell invasion detection, while those without Matrigel coating were used to determine cell migration. 600 μl 10%-FBS containing medium was placed into each bottom chamber, while equal number of suspended cells (1.0–1.5 × 10⁴ cells for migration assay, 3.0–4.0 × 10⁴ cells for invasion assay) in 200 μl medium without FBS were imbedded onto each upper chamber. After cultured at 37 °C with 5% CO2 for 48 h, suspended cells in the upper chamber were washed out, while cells adhering to the bottom membrane were stained by crystal violet. Images were obtained by using the inverted microscope described previously at 100X magnification and cell counting was performed by software Image J.