Millicell chamber

A test to assess HCC cell migration was performed using a MilliCell chamber (12-mm diameter with 8-μm pores; Millipore, Bedford, Massachusetts). Approximately 1 × 10⁵ serum-starved HCC cells were added to the upper chamber in media supplemented with 0.1% FBS, and the chambers were placed into 24-well plates with medium supplemented with 10% FBS. After 24 hours of incubation, a cotton swab was used to scrape the top surface of the transwell chamber. Migrated cells were fixed with paraformaldehyde (4%) and stained with Giemsa solution. The cells were photographed and counted in 5 randomly selected regions under an optical microscope. Labeled cells were counted from images using ImageJ software (https://imagej.nih.gov/ij/).

Cell invasive ability was detected by Matrigel invasion assay using a Millicell chamber (Millipore, Billerica, MA, USA). The Transwell chambers were precoated with 50 µl mixture (Matrigel:serum-free medium 1:5) and the stable clone cell lines (ACHN, 5×10⁴; 786-O, 4×10⁴) in 200 µl serum-free medium were seeded onto the upper chamber for 24 h according to the instructions of the Transwell migration assay. The difference between two groups was analyzed by Sudent's t-test (two-sided).

For cellular migration, 2×10⁴ cells suspended in 200 µl fresh DMEM were plated in Millicell chambers (8-µm pore size; EMD Millipore), and 600 µl DMEM containing 10% FBS was plated in the lower chamber. Cells that migrated through the membrane were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with 0.1% crystal violet for 15 min at room temperature. For cellular invasion assays, the chamber was coated with 50 µl Matrigel for 1 h at room temperature, which was then hydrated for 2 h at 37°C (1:8 dilution; BD Biosciences) prior to the assay. Images of the membrane were captured and the number of migratory or invasive cells was observed under a light microscope (Olympus Corporation) and counted from five fields of view (magnification, ×100).

Cell migration and invasion assays were performed using Millicell^® chambers (8-µm pore size; EMD Millipore). For cell invasion assays, chambers were coated with Matrigel^® (4X dilution; 15 µl/well; BD Biosciences). For cell invasion and migration, cells were transfected with siRNA for 48 h and then seeded in the upper chamber at a density of 1×10⁵ cells/ml in 200 µl of medium containing 0.5% FBS. Medium containing 20% FBS was added to the lower chamber. After 24 or 48 h of incubation, non-invading cells in the upper chamber were removed, and invading cells were fixed in 100% methanol at room temperature for 20 min and stained with 0.5% crystal violet at room temperature for 10 min. Images were captured randomly from 5 fields of each membrane. The number of invasive/migrated cells was counted and the average number of cells per light microscopic field with a magnification of ×200 was presented.