Scratch Assay Tutorial

Create a cell dilution in normal growth media at the appropriate concentration (175,000 cells/mL for this example). Plate 2 mL of the cell suspension into each well of a 6-well plate using a fresh pipette tip for every well to minimize contamination.

Place the seeded plate in the incubator overnight to allow the adherent cells to attach to the bottom of the wells. Keep the plate lid on to minimize contamination from air and external sources.

Carefully aspirate off the normal serum media from each well without touching the bottom to avoid dislodging cells. Replace with fresh low serum media (1% serum for this example) dispensed slowly along the side of each well, using a fresh pipette tip for each well.

Using a 200 µL pipette tip held like a pencil, make a straight scratch across each well from top to bottom while maintaining sterility. The scratch should create clean borders with a cell-free strip down the middle of each well.

Examine scratched wells under the microscope and photograph each well at time zero to establish a baseline. Place the plate back in the incubator for 24 hours to allow cells to begin migration into the scratch.

Photograph wells at 24 hours to observe initial cell migration into the scratch wound. Return plate to incubator and repeat imaging at 48 hours to document continued cell migration and wound closure.

Compare images across timepoints to qualitatively assess cell migration patterns. Optionally use microscope measurements to quantify gap closure and migration rates, then clean up materials.