Scratch Wound Healing Closure Assay

The methods used for a scratch wound healing closure assay have been described previously [25 (link), 26 (link)]. Mouse HF keratinocytes isolated from K5-Cre;PDPN^flox/flox and control mice (at postnatal day 4) were grown to full confluence in 25 μg/ml type IV collagen (collagen from human placenta, Sigma-Aldrich)-coated 24-well plates and were then incubated overnight in serum-reduced medium containing 1% FBS. Cells were scratched with a 200 μL sterile pipette tip and were then incubated. After 48 h, cells were washed with PBS and the images of the scratches were acquired. The surface areas of the cell-free zones were measured and the % scratch closure was determined using TScratch software [26 (link)].