The scratch assay, also known as the wound healing assay, is a straightforward laboratory method used to investigate how cells move and interact in a controlled setting. This technique creates a cell-free gap, or “wound,” in a layer of cells grown in a dish, mimicking a real-life injury. By observing how cells migrate to close this gap over time, scientists gain insights into fundamental biological processes related to cell movement and tissue repair.
The Core Steps of a Scratch Assay
The process begins with cell culture preparation, where cells are grown in a laboratory dish until they form a complete, dense layer, known as a confluent monolayer. Cells are incubated until they reach 75-100% confluence, typically within 18 to 48 hours.
Once the cells have formed a uniform layer, a “scratch” is carefully created across the monolayer. This is often done using a sterile P200 pipette tip, dragged across the cell surface with gentle, consistent pressure to remove cells and create a clear, cell-free zone.
After the scratch is made, the culture medium is aspirated, and the wells are gently washed two to three times with sterile phosphate-buffered saline (PBS) or serum-free medium. This washing step removes detached cells and debris, preventing interference with subsequent cell migration.
Following the wash, fresh culture medium, potentially containing experimental compounds or treatments, is added back to the wells. The cell culture plate is then placed in an incubator, typically at 37°C with 5% CO2, for continuous monitoring. Time-lapse imaging is frequently employed, capturing images of the scratch area at regular intervals, such as every 30 minutes to 2 hours, over 24 to 48 hours.
Maintaining sterile conditions throughout the procedure is important to prevent contamination, which could compromise the experiment. To ensure scratch closure is due to cell migration rather than proliferation, some protocols include steps like overnight serum starvation or the addition of mitomycin C, a DNA synthesis inhibitor, to limit cell division.
Measuring and Interpreting Results
To quantify cell migration in a scratch assay, researchers capture images of the scratch area at different time points using a microscope, often equipped with a digital camera. These images are taken at the same marked locations within each well to ensure consistency.
The closure of the cell-free gap is then measured using image analysis software. Modern methods often utilize specialized software or plugins, such as the “Wound_healing_size_tool” for ImageJ/Fiji, which can semi-automate the quantification of parameters like scratch area, wound coverage, and average scratch width.
By analyzing changes in the scratch area or width over time, researchers determine the rate at which cells migrate to close the gap. A faster rate of closure indicates a higher migratory capacity of the cells under the tested conditions.
Key Applications in Research
The scratch assay is a versatile tool with broad applications in scientific research, offering insights into various cellular behaviors. It is primarily used in studying wound healing mechanisms, allowing researchers to observe how different cell types, such as keratinocytes and fibroblasts, contribute to closing a simulated injury and understanding factors that influence tissue repair.
This assay is also widely used in cancer research to investigate the invasive potential of cancer cells, a process known as metastasis. By observing how cancer cells migrate into the scratch area, scientists can evaluate their metastatic capabilities and test the effectiveness of potential anti-metastatic drugs.
The scratch assay is also valuable for screening and testing the effects of various drugs or experimental compounds on cell migration. Researchers introduce different substances to the culture medium and observe their influence on the speed and extent of wound closure. This application supports drug discovery efforts aimed at modulating cell motility for therapeutic purposes.
Beyond disease-specific studies, the assay contributes to understanding fundamental aspects of cell biology, including how cells interact with each other and their environment during movement. It can also be applied in tissue engineering to study cell-material interactions and optimize designs for regenerative medicine.