8 μm transwell filters

For the transwell migration assay, 5 × 10⁴ PANC-1 cells were plated on 8-μm Transwell filters (Corning). For the invasion assay, 1 × 10⁵ cells were added to the upper chamber of each insert, coated with 150 μg Matrigel (BD Biosciences). The cells were induced to migrate toward medium containing 20% FBS for 24 h (for migration assay) and 48 h (for invasion assay) in the CO₂ incubator. Non-invading cells were removed with a cotton swab. The remaining cells were fixed and stained in dye solution containing 0.1% crystal violet and 20% methanol. The cells that had migrated or invaded were counted and imaged using an IX71 inverted microscope (Olympus Corp.). Ten random fields were chosen, and cell numbers were averaged.
For the scratch wound-healing assay, when the transfected/infected cells reached 80% confluence, a wound was created by scratching with a 200-μl pipette tip. After scratching, the detached cells were removed by washing twice and then the cells were maintained in fresh medium. Cells were imaged at 0 and 24 h after scratching, and the migration distance was calculated by measuring the width of the wound.

The migration and invasion capacities of HTR-8/SVneo and JAR cells were determined with 8 μm Transwell filters (Corning, USA). The transfected cells were resuspended in serum-free medium and added to the upper chambers of the Transwell. Medium containing 20% FBS was added to the lower chambers. In the migration assay, the cells were incubated for 24 hours at 37°C in 5% CO₂ atmosphere and allowed to migrate to the bottom of the well. These cells were fixed with 4% paraformaldehyde and then stained with crystal violet [25 (link)]. In the invasion test, the upper chamber was coated with diluted Matrigel (BD Biosciences, USA), and after 48 hours of culture, the cells were fixed and stained. Five fields of view were randomly selected for observation under the microscope. The experiment was performed in triplicate [27 (link)].

The effect of AR silencing on cell migration was performed using 8-μm transwell filters (Costar, Corning, NY) with modification as described previously 39 (link). For the invasion assay, the upper compartment was coated with 50 μg Matrigel (BD Biosciences, San Jose, CA) to form a matrix barrier. A suspension of cells (20 × 10⁴ per filter for migration and 40 x 10⁴ per filter for invasion) in medium containing 1% FBS and 0.1% BSA was added to the upper compartment. The lower compartment was filled with 400 μl basal medium containing 10% FBS as chemoattractant. After 24 h for migration or 48 h for invasion, the non-migratory cells on the upper surface were removed by a cotton swab and the cells on the lower surface were fixed and stained with the Diff-Quick solution (Dade Behring, Deerfield, Illinois). Migrated or invaded cells in each transwell filter were counted from ten randomly chosen fields. The experiment was performed in quadruplicate and repeated three times independently.