24-Well Cell Invasion Assays, Basement Membrane

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	Culture cells for 12 h in serum-free medium Incubate 48 h for the migration assay and 72 h for the invasion assay

Protocol tips
Culture cells for 12 h in serum-free medium Incubate 48 h for the migration assay and 72 h for the invasion assay

Publication protocol

The migration assay was performed using 24-well Boyden chambers with polycarbonate membranes of 8 μm pore size (Corning Inc., New York, USA). For the invasion assay, the CytoSelect™ Cell Invasion Assay Kit (Cell Biolabs, Inc., San Diego, CA) was used according to the manufacturer's protocol. Cells were cultured for 12 h in serum-free medium. A cell suspension containing 5 × 105 cells/mL in serum-free media containing 0.1% bovine serum albumin (BSA) was prepared; 200 μL was added to the upper chamber and 500 μL media with 20% FBS was added to the lower chamber. After 48 h for the migration assay and 72 h for the invasion assay, the incubation was terminated, and the cells bound to the lower surface of the membrane were fixed with 95% ethanol and stained with crystal violet. The photographs were taken under high magnification with a microscope (Nikon, Tokyo, Japan).

Cell adhesion was determined using the CytoSelect™ 48-well Cell Adhesion Assay Kit (Cell Biolabs) following the recommended protocol. A cell suspension containing 0.1 × 106 cells/mL was prepared in serum-free media, and 150 μL was added to each well. After incubation at 37 °C for 90 min, each well was washed 4 times with 250 μL PBS and incubated with 200 μL crystal violet for 10 min at room temperature. After the cells were washed 4 times with 500 μL deionized water, the wells were air-dried for 30 min.

For quantitative analysis of each experiment, the crystal violet was dissolved in 200 μL extraction solution per well, and the absorbance was measured at 590 nm in a plate reader (Bio-Rad, Hercules, CA, USA). All experiments were done in triplicate.

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