Wound healing assay

A wound healing assay, also called a scratch assay, is a lab method for studying how cells move and interact with each other. It works by creating a scratch—through mechanical, thermal, or chemical damage—across a layer of cells grown in a dish, then using time-lapse microscopy to capture images at set intervals. This is a two-dimensional approach that semi-quantitatively tracks how a sheet of cells migrates to close the gap. It works best for cells that move together in epithelial sheets and is not suitable for non-adherent cells or for studying chemotaxis.

The technique has several strengths. It is inexpensive, simple to perform, and allows real-time observation. Experimental conditions can be easily adjusted for different goals, and the strong directional movement makes data analysis straightforward. However, there are drawbacks. Manual scratching can lead to inconsistent scratch depth and width, as well as ragged edges that complicate analysis. The damage may also harm cells near the wound, skewing wound size measurements. Automated tools are reducing these issues: Electric Cell Impedance Sensing avoids damaging the underlying matrix, and the Woundmaker creates fast, uniform wounds across 96- or 384-well plates, enabling high-throughput screening for medical research. Even with better technology, confounding factors remain, such as cell crowding, cell-cell adhesion effects, matrix influences, and uneven cell density at the scratch edge. Critics also note that a scratch is a poor mimic of a real wound, which is far more complex, though the assay still offers useful insights into collective cell movement under controlled conditions. Despite its apparent simplicity, the method has been criticized for inconsistent application between experiments.

A standard protocol without advanced tools goes like this: Plate cells in growth medium in a live-cell imaging dish or chamber slide, ensuring a uniform monolayer—clumps cause inaccurate results, so titrate to find the optimal density. Once cells are confluent, use a pipette tip to scratch a wound across the center of the well. For manual scratches, the wound should be about 0.5 mm wide and visible on both sides of the field of view.