Millicell tissue culture plate well inserts

Cell migration was analyzed using a wound-healing and transwell-migration assay. For the wound-healing assay, infected cells were cultured in six-well plates. When the cells were up to 90% confluence, three scratch wounds across each well were made using a P-200 pipette tip. Plates were washed twice with PBS to remove detached cells and incubated with the complete growth medium without FBS, and the wound-closing procedure was observed for 48 h. Photographs were taken at 0, 6, 24, and 48 h, respectively. The result was expressed as a migration rate: the area covered by the migrating cells (time)/the wound area (0 h). For the transwell-migration assay, cells were serum-starved for 6 h and suspended in serum-free RPMI 1640. The cell suspension (5 × 10⁴ cells) was added to the upper chamber of Millicell^® tissue culture plate well inserts (Merk Millipore, Billerica, MA, USA) without Matrigel, and was incubated for 24 h with medium containing 10% FBS in the bottom of the chamber. Residual cells on the upper side of chambers were removed by scraping with cotton swabs, and the cells that attached to the lower side of the membrane were fixed with methanol for 10 min and stained with 0.4% crystal violet/50% methanol for 10 min. Cell numbers were counted in four random fields (×100) per filter and photographs were taken by microscopy.

For the Transwell-migration assay, AGS cells were treated with2,4-DAQ (0 to 300 μM) for 24 h then were serum-starved for 6 h. Then, 5 × 10⁴ cells were seeded onto the upper chamber (8-μm pore size) of Millicell^® tissue culture plate well inserts (Merk Millipore, Billerica, MA, USA) without Matrigel and incubated for 24 h with medium containing 10% FBS in the bottom of the chamber. Residual cells on the upper side of chambers were removed by scraping with cotton swabs and the cells that attached to the lower side of the membrane were fixed with methanol for 10 min and stained with 0.4% crystal violet/50% methanol for 10 min. The invaded cells were counted in four random fields (×100) per filter, and photographs were taken by microscopy. For the invasion assay, the Matrigel-coated chambers purchased from BD Biosciences were used. Protocols were similar as described in the Transwell-migration assay.