Scratch wound healing assay

The scratch wound healing assay was performed to determine the effects of MFSOL on the proliferation and migration of human keratinocytes. These cells were dispersed in DMEM with 10% FBS (5×10⁴ cells/mL) and incubated with 5% CO₂ at 37°C in a 24-well plate. When the cells formed a confluent monolayer, they were scratched using a vertical tip with 200 μL on each well. Cell debris was removed and the wells were washed with phosphate buffered saline (PBS). Photomicrographs (200×) were taken after 24, 48, and 72 h of incubation with MFSOL (25, 50, and 100 μg/mL) in 1 mL of fresh medium (16). To evaluate only the migration, an antimitotic agent, mitomycin C (10 μg/mL; Sigma®, USA), was added for 1 h, following the scratch protocol. Immediately thereafter, the cells were treated with MFSOL (25 and 50 μg/mL) and, after obtaining photomicrographs, the migration was evaluated at the initial time and after incubation for 24 and 48 h (17). The open area of the scratch was measured using the TSCRATCH® software (ETH Zurich, CSE Lab, Switzerland) in each analysis period at the same initial site. The percentage of open area was measured using the formula: Open area (%) = open area at time X / open area at initial time × 100.