Directional Collective Migration in Wound Healing Assays

Cell migration is suppressed by confluence in a process called contact inhibition. Relieving contact inhibition upon scratching is one of the simplest ways to induce cell migration in a variety of cell types. Wound healing is probably most relevant to epithelial monolayers, because epithelial cells generally assume a barrier function, which must be restored as fast as possible by the healing process. This versatile assay, however, can also be applied to fibroblasts and to tumor cell types. Furthermore, assessing the cell response to scratch wounding requires no special equipment or reagents. It is one of the few cell migration assays, which can even be performed without videomicroscopy, since the closure of the wound can be estimated at fixed time points. Several hours after wounding, directional collective migration is easily assessed and quantified. However, cell proliferation, which is also induced by the relief of contact inhibition, is one of the confounding factors of wound healing assays that must be taken into account. A recent alternative to the scratch-induced wound is to use special inserts to seed cells into closely spaced chambers. When the insert is removed, contact inhibition is relieved, similar to the scratch-induced wound. In this chapter, we provide the protocol of the two methods and compare their advantages and disadvantages. We also provide a protocol to estimate cell proliferation upon wound healing based on the incorporation of the nucleotide analog EdU.