The scratch assay, often referred to as a wound healing assay, is a widely used laboratory technique. It investigates cell migration, the directed movement of cells. This method involves creating a gap in a layer of cells and then observing how those cells move to close the created space. This approach is valuable for studying how groups of cells coordinate their movement.
How the Scratch Assay Works
Performing a scratch assay begins with preparing a cell monolayer, a single, confluent layer of cells grown on a cell culture dish. Cells are seeded at a density that allows them to reach 95-100% confluence within 24 to 48 hours, forming an even, continuous sheet. Avoid shaking the culture vessel during this initial growth phase to ensure uniform cell distribution.
Once the cells have formed a confluent monolayer, the next step involves creating a “scratch” or cell-free gap. This artificial wound is made by gently scraping the cell layer with a sterile pipette tip or a specialized tool. The pipette tip is held perpendicular to the well bottom and dragged across the monolayer with consistent pressure and speed to create a straight line. Multiple parallel scratches or a cross shape may be made to increase the observed area.
After the scratch is made, the culture medium is removed, and the monolayer is washed with PBS to remove any detached cells. Fresh culture medium, which may contain test compounds, is then added. The culture plate is placed onto a microscope stage, often within a live-cell incubator, to begin monitoring.
Images of the scratch are captured at regular intervals, such as every 2, 4, 6, or 12 hours, for the duration of the experiment. Phase-contrast microscopy is used to visualize the cell-free gap and the cells at its edges. Consistent focus and lighting are maintained, and reference points are marked to ensure the same region is imaged repeatedly over time.
What a Scratch Assay Reveals
The scratch assay provides insights into cellular behavior, particularly cell migration. Researchers use this assay to understand cell movement, including parameters like migration speed, persistence, and polarity. The technique is well-suited for studying how cells collectively migrate, mimicking processes seen in the body.
This assay is applied to investigate wound healing processes, as the created scratch simulates tissue damage. By observing how cells migrate to close the artificial wound, researchers gain insights into the mechanisms of tissue repair and re-epithelialization. This understanding contributes to advancements in treating non-healing wounds.
The scratch assay is also used in cancer research to assess the metastatic potential of cancer cells. The ability of cancer cells to migrate is a key step in tumor metastasis, making this assay relevant for evaluating how invasive different cancer cell lines are. It also helps in screening compounds that influence cell motility, supporting drug discovery for anti-metastatic treatments and other therapeutic interventions.