Transwell matrigel invasion chambers

Wound healing experiments were conducted to evaluate the migration capability of cells. Cells were inoculated on 6-well plates and allowed to grow from 80 to 90% confluence. Then, cell monolayers were wounded by a 200-µl pipette tip. The migration distance of the cells was quantified at 0 and 48 h. Migration experiments were performed in 24-well Transwell Matrigel invasion chambers (8 μm pores; BD Biosciences) based on the manufacturer’s instructions. In general, cells were starved for 12 h, and then 4.5×10⁴ cells were inoculated into the upper chambers. The lower chambers were filled with DMEM with 15% FBS. After 1 day, the migrated cells were counted.

The invasion assays were performed with the Transwell Matrigel Invasion Chambers (BD Biosciences. Bedford, MA, USA). Following 24 h serum starvation, trypsinized cells (5 × 10⁴ cells /well) were resuspended in DMEM containing 0.1% FBS and added to upper chamber of transwell inserts. DMEM supplemented with 10% FBS was used in the lower chamber to act as a chemoattractant. After 24 h, cells that had migrated through the membrane were fixed and stained with the HEMA 3 staining kit (Fisher Scientific, Pittsburgh, PA, USA). The invaded cells in four random fields were counted and expressed as the average number of cells per field under light microscopy (Eclipse TE2000, Nikon, Tokyo, Japan).

Stably transduced 4×10⁴ cells were added to matrigel transwell invasion chambers or control transwell chambers (BD Biosciences) and incubated for 72 h with chemoattractant media (Clonetics, Walkersville, MD, USA) supplemented with growth factors. Cells invading through the matrigel or control membranes were fixed using 70% ethanol, stained with 0.1% crystal violet, and photographed in 4 fields to cover the entire area. Cells were counted from all fields by a scientist blinded to the experimental conditions. For colony formation assay, 1.0×10⁴ cells were plated into 6-cm dishes in 2 ml of medium containing 0.3% agarose, overlaid with 2 ml of 0.5% agarose. Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, Waltham, MA, USA) was added on top, and dishes were maintained in a humidified atmosphere with 5% CO₂ at 37°C for 12 days. Then colonies were stained with crystal violet, and the number of colonies was counted.

Cells (4 × 10⁴) were added to Matrigel transwell invasion chambers or control transwell chambers (BD Biosciences, San Jose, CA) and incubated for 24–72 h with chemoattractant media (Clonetics, Walkersville, MD) supplemented with growth factors. Cells invading through the Matrigel or control membranes were fixed using 70% ethanol, stained with 0.1% crystal violet, and photographed in four fields to cover the entire area. Cells were counted from all fields by a scientist blinded to the experimental conditions.