2.3. Scratch Wound Healing Assay

Human primary keratinocytes experiencing three passages were used in experiments and the seeding density was fixed at 10,000 cells/cm² in a 12-well culture plate, which was determined by the trypan blue exclusion test on a hemocytometer. The cells were allowed to grow until confluence for 72 h with medium changed at 48 h. Upon confluency, scratch was introduced using a 10 µL pipette tips and the cells were washed with a warm Dulbecco phosphate buffer saline (DPBS). The cells were treated with EpiLife™ medium with or without TGFβ or KH. The 12-well culture plate was mounted on automated stage of the light microscope. Picture of the cell-free area were taken every 15 min for 24 h. The leading edges of the migrating cell population were manually tracked and quantified with validated image analysis software, ImageJ [23]. Percentage of wound area reduction was calculated where the wound area at 0 h is considered as 100%.