2.5. Cell Scratch Wound Healing Assay

The cell scratch wound assay is a straightforward and economical method to study keratinocyte migration collectively in vitro [23]. HaCaT cells uninfected or infected with recombinant adenoviruses or siADAM17 were grown to confluence in 24-well plates in serum-conditioned RPMI 1640. Scratch wounds were created in the confluent monolayers using a sterile p200 pipette tip, and different fields were captured under normoxic or hypoxic conditions. Four perpendicular semiopaque marks were placed across each scratch on the external surface of the well to standardize the quantitative analyses. After the adherent cells were washed three times, the wounded monolayers were cultured in RPMI 1640 medium. After incubation for the indicated hours, the repopulation of the wounded areas was observed under a Zeiss imaging system. Using the NIH ImageJ image processing program, the size of the denuded area was determined at each time point from the digital images.