6 well flat bottom microtiter plates

To investigate fibroblast migration, cells were plated on 6-well flat bottom microtiter plates (Thermo Fisher Scientific, Waltham, MA USA) and grown in 2 ml growth medium. Once about 90% confluence was reached, medium was removed and a straight scratch along the monolayer was created in the centre of the well using a sterile P-200 pipette tip, as described elsewhere [34 (link)]. Cellular debris was gently removed with Dulbecco’s phosphate-buffered saline (PBS) and cultures were exposed to undiluted extracts. Images of wound closure were obtained at 0, 18, 24 and 48 h, using a conventional phase-contrast microscope (Olympus, Tokyo, Japan). Photographs were taken at 200× magnification to obtain cell behavior profiles of migration and morphology.

To investigate the effect of DM2 on keratinocyte migration, cells were plated into 6-well flat bottom microtiter plates (Thermo Fisher Scientific, Waltham, MA, USA), cultured in DMEM supplemented with 7% FBS and incubated at 37 °C in a humidified 5% CO₂ atmosphere. At about 90% confluence, medium was removed, and the adherent cell layer was scratched using a sterile P-200 pipette tip, as described elsewhere [41 (link)]. Cellular debris was gently removed with PBS washing. Then, monolayers were exposed to different concentrations of DM2 (0.1, 1.0, and 10 µM) and cells were grown at 37 °C in humidified 5%, CO₂ atmosphere. Control cells received only fresh DMEM. At 0, 16, 32, and 48 h after treatment, the monolayers were washed twice with PBS, fixed with 4% paraformaldehyde at 4 °C for 30 min, and rinsed with PBS. Cells were stained with 1% crystal violet solution for 10 min at room temperature. Then, cells were washed three times in PBS, and the images were captured at 0, 16, 32, and 48 h using a conventional phase-contrast microscope (Olympus, Tokyo, Japan). The wound closure areas were calculated by ImageJ. Photographs at 200× magnification provided migration and morphology profiles.