Matrigel coated transwell insert

The cells are seeded on 6-well plate, and when the cells are 80% confluent, a cell wound is created by scraping the cells with the tip of a micropipette. The medium was then changed immediately, and spontaneous cell migration was monitored at 0 and 48 h using a microscopy (Olympus, Japan).
Cell migration and invasion assays.
The migration and invasion abilities of cells were assessed using non-Matrigel-coated and Matrigel-coated Transwell inserts (BD Biosciences, San Diego, CA, USA), respectively. For cell migration, 5 × 10⁴ cells were added to the upper chamber, fibronectin-free medium was added to the lower chamber, and the cells were incubated at 37 °C for several hours. For cell invasion, 1 × 10⁵ cells were added to the upper chamber, medium containing fibronectin (20 μg/mL) was added to the lower chamber, and the cells were incubated at 37 °C for several hours. Migrated or invaded cells (on the lower side of the membranes or in the lower well) were then fixed in 4% paraformaldehyde for 20 min and stained with hematoxylin for 15 min. The cells were counted under a microscope.

MDA-MB-231 cells were serum-starved overnight prior to plating in invasion assays. Trypsinized cells were plated into the upper chamber of control (8 μm pore) or Matrigel-coated Transwell inserts (BD Biosciences) containing serum-free DMEM-Hi medium at a density of 10,000 cells/well and attracted to medium containing 10 % FBS. Cells were allowed to migrate/invade for 24 h at normoxia or hypoxia (0.5 % O₂) (n ≥ 3 wells per genotype per condition). To compare invasion of CD49f-FITC-sorted populations, cells were collected post-sorting into serum-coated FACS tubes as described above, washed once with PBS and then allowed to recover from sorting overnight at 4 °C in tumorsphere stem cell media, as in [3 (link)]. Cells were then plated at a density of 30,000−50,000 cells/well and exposed to hypoxia (0.5 % O₂) for 24 to 48 h (n ≥3 wells/population/experiment). Crystal violet stained filters were imaged using ImageJ software and the invasion index calculated following correction for random migration per manufacturer's instructions.

For assessing cell migration, 15,000 cells in serum-free medium were seeded into Transwell inserts (353097, Corning) containing 8-μm permeable pores and allowed to migrate toward 10% FBS-containing medium. After 16 h, the Transwell inserts containing the cells were removed and washed in PBS three times. The migrated cells on the bottom of the insert were fixed with 5% glutaraldehyde solution followed by crystal violet (1%) staining. After washing the inserts three times with water, the inserts were allowed to air dry, and pictures were taken using an inverted microscope. Ten independent fields were counted for each Transwell, and the average numbers of cells/field were represented in graphs. For assessing cell invasion, 15,000 cells in serum-free medium were seeded into the Matrigel-coated Transwell inserts (BD Biosciences). After 16 h, the invaded cells were stained, counted, and represented in a graph similarly to the migration assay protocol.

The migration and invasion abilities of pancreatic cancer cells were evaluated using non-Matrigel-coated and Matrigel-coated Transwell inserts (BD Biosciences, San Diego, CA, USA), respectively. Briefly, 5×10⁴ cells in 500 μl of serum-free medium were added to the upper chamber, and the medium containing 10% FBS was added to the lower chamber. The cells were left to invade the Matrigel-coated for 48 h, and non-Magrigel-coated for 24 h. The non-invading cells on the upper surface of the membrane were removed by wiping, and the invading cells were fixed and stained with crystal violet. The number of migrating or invading cells was counted under a microscope in five predetermined fields for each membrane at ×200 magnification.

The invasive potential of BE (2)-C cells in vitro was measured by evaluating the number of invading cells using Matrigel-coated trans-well inserts (BD Biosciences) according to the manufacturer's instructions. BE (2)-C cells transfected with the indicated shRNA were seeded onto an insert containing 8 µm pores (BD Biosciences) in a 24-well plate at 1×10⁵ cells/ml. Cells on the lower side of the membrane were fixed with 4% paraformaldehyde and stained using a Diff Quick Staining Kit (Sysmex).