Introduction to the Cell Scratch Assay

I. Experimental Principle

    The cell scratch (wound healing) assay is a simple method for assessing cell migration and repair capabilities. Similar to an in vitro wound healing model, a line is drawn in the central region of a monolayer of adherent cells cultured in a dish or plate using a micropipette tip or other hard object to remove the cells in that area. The cells are then cultured for the duration specified by the experiment. Afterward, the culture plate is removed, and the growth and migration capabilities of the surrounding cells are observed. The experiment typically requires the establishment of a normal control group and an experimental group. The experimental group is treated with a specific factor, drug, or exogenous gene. By comparing the differences in wound-healing capacity among the various groups, the migration and repair capabilities of the cells in each group can be assessed.

II. Experimental Procedures

1. All equipment must be sterilized. Rulers and marker pens should be exposed to UV light for 30 minutes prior to use.

2. Using a ruler as a guide, draw lines with a marker pen on the back of a 6-well plate, approximately every 0.5–1 cm, across the wells. Draw at least 5 lines per well.

3. Seed approximately 5 × 10⁵ cells (the exact number varies by cell type; adjust so that the wells are completely covered overnight) and incubate in a 37°C incubator.

4. The next day, using a 10 µl pipette tip aligned with the ruler, draw two parallel lines perpendicular to the marked lines. Keep the pipette tip vertical; do not tilt it.

5. Wash the cells three times with PBS to remove the scratched cells, then add serum-free medium.

III. Precautions

1. When washing with PBS, add the solution slowly and carefully to avoid dislodging the adherent monolayer, which could affect the experimental imaging results.

2. Scratch assays are typically performed using serum-free or low-serum (＜2%) media; otherwise, cell proliferation cannot be ignored. (Alternatively, treat with mitogen for one hour.)

3. Make the scratch along the vertical direction of the lines drawn on the back of the 6-well plate to form a fixed intersection point, which serves as a fixed reference point to resolve the issue of inconsistent positioning during observations at different times.