Biocoat matrigel invasion chambers with 8 μm pores

Cells either stably (H520) or transiently (H1299) transfected with DLK1 or null vector were cultured separately until 80% confluence. The cells were then washed three times with phosphate-buffered saline (PBS) and cultured in serum-free media overnight before being subjected to an ECM invasion assay in vitro. The chemoinvasion assay was conducted using BioCoat Matrigel Invasion Chambers with 8 μm pores (BD Biosciences, Bedford, MA) according to the manufacturer's instructions. Briefly, 2.5×10⁴ cells were resuspended in fresh serum-free media and seeded into the upper chamber of a 24-well transwell plate, while the lower chamber contained fresh culture media with 20% FBS as a chemoattractant. The cells were allowed to invade for 22 hours (37°C, 5% CO₂ atmosphere), and the chambers were then washed with PBS. Those cells that did not invade through the membrane were removed. The invading cells on the lower surface of the membrane were fixed with cold methanol, stained with 0.2% crystal violet and examined. The cells on each membrane were counted in no less than five fields under a light microscope.

The transwell invasion assay was performed with Biocoat™ Matrigel™ Invasion Chambers with 8-μm pores (BD Biosciences) as directed by the vendor’s protocol. AGR2-knockdown SNU-478 or AGR2-overexpressing SNU-869 cells (5 × 10⁴ cells/well) were resuspended in 200 μL serum-free RPMI medium and then added to the upper chamber. Bottom chambers were filled with 1% fetal bovine serum-RPMI, and the cells were incubated for 24 h at 37°C in a 5% CO₂ incubator. Cells present in the coated membrane were fixed with 3.7% formaldehyde in PBS, permeabilized with 100% methanol for 20 min, and stained with 1% crystal violet for 1 h. The membrane was detached, wiped with a cotton swab, and examined under the microscope at 100 × magnification (Olympus, Tokyo, Japan).

Cell invasion experiments were performed using BioCoat Matrigel Invasion Chambers with 8 μm pores (BD Biosciences, Bedford, MA, USA), according to the manufacturer’s instructions. Briefly, cells were seeded at 2.5×10⁴ cells per well in serum-free medium overnight, and subsequently added to the upper chamber of a 24-well transwell plate. The lower chamber contained fresh culture media with 20% FBS as a chemoattractant. Cells were allowed to invade for 24 h at 37°C in the 5% CO₂ atmosphere, and the chambers were then washed with PBS. Cells that did not invade through the membrane were removed, while the invading cells on the lower surface of the membrane were fixed with cold methanol, stained with 0.2% crystal violet and examined. The number of invading cells in each chamber was counted in at least five fields under a light microscope (OLYMPUS CKX41; Olympus Corp., Tokyo, Japan).