Transparent pet membrane

For assessment of Transwell migration, cell culture inserts (Corning, 353097) with 8.0 µm Transparent PET Membrane, were each placed into a 24- well plate with 800 μL complete growth medium (with serum) added at the bottom of each well. 50,000 and 30,000 cells of OVCA433 and SKOV3, respectively were seeded onto the top of the Transwell membrane of the insert in 150 μL serum free media. After 24 hours, the Transwelll inserts were removed and washed twice with 1XPBS. The Transwell membrane was fixed and stained with Crystal violet (0.05%) for an hour. The inserts were washed three times with 1X PBS and the non-migrated cells were removed from the top of the membrane using dry cotton swabs. The inserts were dried overnight, and images were taken of the migrated cells at the bottom of the membrane using Leica Thunder Imager with colored K3C camera. The dye was released from cell inserts using 30% acetic acid and absorbance was measured using GloMax Explorer (Promega) microplate reader at 590 nm as a readout for cell migration.

Cells were transfected for 24 h and plated in the upper chamber of the transwell in 24-well plates (8.0 µm Transparent PET Membrane, Corning, NY, USA) at 1×10⁵ cells in 200μL of serum-free medium per chamber and for invasion there was Matrigel (BD Pharmingen) added into the upper chamber, 500 μL RPMI-1640 medium with 15% FBS were added into the lower chamber as a chemoattractant. After 48h, cells in the upper membrane were removed completely and 4% paraformaldehyde fixed for 15 minutes, then 1% crystal violet stained for 10 minutes, pictures were taken under the inverted microscope (NIKON, Japan).

For invasion assays, a Falcon Permeable Support was added to a six‐well plate with 15 μL 5 mg/mL Matrigel Matrix (Corning) on an 8‐μm Transparent PET Membrane (Corning) 48 h after seeding. The infiltrated cells were stained with Diff‐Quik (JACLaS) on the Transwell membrane surface, counted in five randomly selected fields at ×200 magnification, and quantified using Image J software (https://imagej.net/). Cells were seeded in a six‐well flat‐bottomed plate and incubated until 90% confluency. Confluent cell monolayers were scratched using plastic tips. Images of scratched areas were captured 24 h after scratching and evaluated as the percentage of migration (migration index: 1−[length at 24 h]/[length at 0 h]). Cells were seeded in six‐well flat‐bottomed plates in RPMI supplemented with 10% FBS. After 10 days of culture, colonies were fixed and stained with Diff‐Quik. The number of colonies was counted using Image J software (https://imagej.net/).

The cell invasiveness assay used Falcon Permeable Support for 24‐well plate with 15 µL of 5‐mg/mL Matrigel Matrix (Corning Inc., Corning, NY) on an 8.0‐µm Transparent PET Membrane (Corning Inc.). Stained cells using Diff‐Quik (JACLaS, Tokyo, Japan) on the transwell membrane surface were counted in five randomly selected fields at ×200 magnification and quantified using Image J software (https://imagej.net/). Huh7 and Hep3B cells were seeded in six‐well plates and incubated until 90% confluent. Confluent cell monolayers were scratched by plastic tips. Images of scratched areas were captured over the following 24 hours, measured using Image J software and evaluated as percentage of migration (migration index: 1 – [length at 24 hours]/[length at 0 hours]). In addition, Huh7 and Hep3B cells were added to each well in six‐well flat‐bottomed plate in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS. After 10 days of culture, the small population of cells were fixed using 4% paraformaldehyde and stained using crystal violet. The number of colonies was counted. These experiments were repeated three times.