Millicell invasion chamber

The cell invasion assay was performed as previously described [35 (link),36 (link),37 (link)]. Briefly, 5% Matrigel (Becton Dickinson Biosciences, Bedford, MA, USA) was used to coat the membrane of the upper insert of a Millicell invasion chamber (Millipore, Burlington, MA, USA) with a pore size of 8 μm in a 24-well plate. Cells in a medium containing 1% FBS were seeded into the upper insert. The lower chamber contained a medium with 20% FBS to trap invading cells. After 16 to 24 h of incubation at 37 °C, the invasive cells were determined by observing the reverse side of the upper insert after being fixed with formaldehyde and stained with crystal violet.

The cell migration ability was determined by using the in vitro wound-healing assay (8 (link), 9 (link)). Briefly, cells were seeded in an ibidi^® culture insert (Applied BioPhysics, Inc. NY) on top of a 6-well plate. After 8 hr of incubation, the culture insert was detached to form a cell-free gap in a monolayer of cells. After changing to culture medium with 1% FCS, the cell migration status toward the gap area were photographed with a specific period time point. The cell invasion ability was evaluated by using the BioCoat Matrigel (Becton Dickinson Biosciences, Bedford, MA) and Millicell invasion chamber (Millipore Corporation, Bedford, MA) (8 (link), 9 (link)). The Matrigel were fist coated onto the membrane of the Millicell upper chamber with a pore size of 8 μm in a 24-well plate. Cells in 1% FBS medium were seeded into the upper chamber. The lower chamber will contain 10% FBS in medium to trap invading cells. After a specific time point, the cells invading to the reverse side of the membrane were fixed, stained, and photographed.

Cell invasion ability was evaluated by using a BioCoat Matrigel (Becton Dickinson Biosciences, Bedford, MA, USA) and Millicell invasion chamber (Millipore Corporation, Bedford, MA, USA) [26 (link),27 (link)]. The Matrigel was first coated onto the membrane of the Millicell upper chamber with a pore size of 8 μm in a 24-well plate. Cells in 1% FBS medium were seeded into the upper chamber. The lower chamber will contained 10% FBS in medium to trap invading cells. The invasion ability was determined by observing the reverse side of the upper chamber after being fixed and stained with crystal violet.

The transwell invasion assay was performed using a Millicell invasion chamber (Millipore, Billerica, MA, USA). The 8-μm pore inserts were coated with 15 μg of Matrigel (Becton Dickinson Labware, Bedford, MA, USA), and 5×10⁴ cells were seeded in the top chamber. The Matrigel invasion chamber was incubated for 24 h in a humidified tissue culture incubator. Non-invading cells were removed from the top of the Matrigel with a cotton-tipped swab. Invading cells on the bottom surface of the filter were fixed in methanol and stained with crystal violet. Invasion ability was determined by counting the stained cells.

The invasive abilities of the cells were determined by culturing the cells on a polycarbonate membrane coated with Matrigel (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA) in a Millicell invasion chamber (Millipore, Billerica, MA, USA) as previously described
[23 (link)]. Briefly, Matrigel was first coated onto the membrane of the Millicell upper chamber for 12 h at 37°C. After transfection of the miR-196 overexpression plasmids or antagomir oligonucleotides, the cells were seeded in the upper chamber with 1% FBS medium. The lower chamber contained complete culture medium (containing 10% FBS) to trap invading cells. After incubation at 37°C, the cells that invaded through the Matrigel-coated membranes into the lower chamber were stained with crystal violet and photographed. All the experiments were performed at least three times independently and that typical results were shown. In each sample, the invasion ability was quantified by comparing the density of crystal violet dye after normalization to the control group. The error bars shown in the relevant figures indicated the standard deviation of the quantification results in all experiments.