2.14. Migration Assays

Transwell permeable supports with a 6.5 mm polycarbonate membrane and 8 µm pores were used to separate the upper and lower chambers of a 24-well plate. Both sides of the membrane were coated with 1 µg/mL human plasma fibronectin (Sigma, Saint Louis, MO, USA). Bovine fibronectin was depleted from the complete media using gelatin Sepharose (GE Healthcare, Salt Lake City, UT, USA) following the manufacturers protocol. Complete media containing 10 ng/mL TGF-β1 was then added to the lower chamber at 600 µL/well. Cells were trypsinized, pelleted, resuspended in low serum media containing 0.5% FBS and added into the upper chamber at 1.0 × 10⁴ cells/well in 100μL media per well. After allowing attachment, antibody treatment, rPro or PBS blank was added to the lower chamber and the plate was incubated for 5 h at 37 °C, 5% CO₂. Media was aspirated, and cells were fixed using 4% Formaldehyde for 15 min at room temperature. Cells were removed from the top of the upper chamber using a sterile cotton swab and wells were washed three times with PBS. Adherent cells on the apical side of wells were then stained by applying DAPI solution (Sigma, Saint Louis, MO, USA) following the manufacturers protocol. Nuclei were visualized by fluorescent microscopy and counted using the Biotek Cytation 3 and latest Gen5 v3.11 software (Winooski, VT, USA). Samples were imaged using the 2.5× objective and total cell numbers were counted in each frame.