Matrigel coated transwell cell culture chambers

The cell invasion assay was performed using Matrigel-coated transwell cell culture chambers (Corning, MA). Normal or siRNA transfected proliferating Caco-2 cells were collected and resuspended at a density of 1 × 10⁵ cells/ml in FBS-free media. 250 μl of cell suspension were added to the upper chamber and 500 μl of 10% (v/v) FBS in MEM medium added to the lower chamber. Cells were incubated first with or without ECG or EGCG dimers for 30 min, and subsequently with or without EGF (10 ng/ml) for 48 h. All additions were made to the upper chamber. Subsequently, the medium in the upper chamber was removed, transwell inserts were washed twice with PBS, cells fixed with paraformaldehyde (3.7% (w/v) in PBS) for 2 min and permeabilized in 100% methanol for 20 min. Cells in the upper chamber were removed using a cotton swab and the transwell chambers air dried. Invasive cells migrating to the back of the membrane were stained with 0.1% (w/v in PBS) crystal violet. The invasiveness of Caco-2 cells was defined as the total number of cells in 3 randomly selected microscopic fields.

To compare the invasive ability between the cells transfected with siRNAs targeting PD-L1 and those transfected with a non-targeting siRNA, an invasion assay was performed using Matrigel-coated Transwell cell culture chambers (8 μm pore size; Corning Incorporated, Corning, NY, USA). The cells were cultured in a serum-free medium and resuspended. Subsequently, 40 μL of the cell suspension was coated onto the membrane of the Transwell plate and air-dried for 3 h. Next, 5 × 10⁴ cells were added to the serum-free medium and placed in the upper chamber. The medium (600 μL) containing 10% FBS was added in the lower chamber, and plates were incubated at 37 °C in a 5% CO₂ atmosphere for 24 h and 48 h. Cells that did not penetrate the membrane in the upper chamber were removed using a cotton swab. Those on the lower surface that passed through the Matrigel and membrane were fixed with 4% formaldehyde and later stained with 2% crystal violet in 2% ethanol. Cells were imaged and counted under a light microscope at 200× magnification using three randomly selected sections from each membrane.