Wound Healing Assay for Migratory Capacity

To determine the migratory capacity of the treated cells, wound healing assays were performed^{49 (link)} after treatment with IC₅₀ concentrations alone with or without EP. After treatment, 4 × 10⁴ cells were plated on a 24-well plate with a silicone insert (Ibidi) that formed a 500-μm ± 50 μm cell-free gap after removal. Cells were incubated overnight at 5% CO₂ and 37 °C until a confluent cell monolayer was formed, then the silicone inserts were removed. Images of the wounds were captured with DP72 CCD camera connected to Olympus IX-70 inverted microscope at 0 h (when culture inserts were removed) and then every 2–4 hours (depending on the cell line used and optimized in preliminary tests), until the wounds of the control group were completely sealed (for HUVEC 12 h, CHO 24 h, FaDu 26 h). At each time point, the cell-free area was quantified using FIJI image analysis software^{50 (link)} and a kinetic analysis was made from the obtained values. The cell migration rate of each experimental group was normalized to the migration rate of untreated cells. The assay was not performed in B16F1 cells because these cells have very limited migratory potential.