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Scratch Wound Healing Assay

Cancer Biology > Metastasis
Author: Yanling Chen, 8/5/2011, 23339 views, 5 Q&A

[Abstract] Scratch Wound Healing Assay has been widely adapted and modified by researchers to study the effects of a variety of experimental conditions, for instance, gene-knockdown or chemical compound treatment, on cell migration and proliferation. The assay is simple and inexpensive, and the experimental conditions can be easily modified for different purposes. The basic principle of the assay is that, a “wound gap” in a cell monolayer is created by scratch, followed by monitoring the “healing” of this gap by cell migrating and growth towards the center of the gap, hereby filling up the “gap”. Factors that alter the motility and/or growth of the cell can lead to increased or decreased rate of “healing” of the gap (1). The assay is quantitative, and is suitable for high-throughput screen if antomated system is used (2).

Materials and equipments

  1. Human MDA-MB-231 cell (ATCC #HTB-26)
  2. Dulbecco's Modified Eagle Medium, DMEM (Invitrogen #10313-021)
  3. Fetal Bovine Serum (ATCC #30-2020)
  4. PBS (Invitrogen #14190-144)
  5. BD Falcon 24-well tissue culture plate (Fisher #08-772-1H; BD #353226)
  6. Rainin pipet tips, 1 ml (Rainin #GPS-L1000)
  7. Glutaraldehyde (Sigma-Aldrich #G6257)
  8. Ethanol (Sigma-Aldrich #459836)
  9. Crystal violet (Sigma-Aldrich C3886)
  10. Cell culture incubator: 37 °C and 5% CO2.


  1. Cells were grown in DMEM supplemented with 10% FBS.
  2. Cells were seeded into 24-well tissue culture plate in a density that, after 24 hours of growth, they should reach ~70-80% confluence as a monolayer.
  3. Do not change the medium. Gently and slowly scratch the monolayer with a new 1 ml pipette tip across the center of the well. While scratching across the surface of the well, the long-axial of the tip should always be perpendicular to the bottom of the well. The resulting gap distance therefore equals to the outer diameter of the end of the tip. The gap distance can be adjusted by using different types of tips. Scratch a straight line in one direction.
  4. Scratch another straight line perpendicular to the first would line to create a cross in each well.
  5. After scratching, gently wash the well twice with medium to remove the detached cells.
  6. Replenish the well with fresh medium.
  7. (Medium may contain ingredients of interest, e.g., chemicals that inhibit/promote cell motility and/or proliferation.)
  8. Grow cells for additional 48 hours (or the time required).
  9. Wash the cells twice with 1x PBS then fix the cells with 3.7% paraformaldehye for 30 minutes.
  10. Fixed cells are stained with 1% Crystal Violet in 2% ethanol for 30 minutes.
  11. Take photos for the stained monolayer on a microscope. Set same configurations of the microscope when taking pictures for different views of the stained monolayer. The gap distance can be quantitatively evaluated usingsoftwares like Photoshop or ImagJ (http://rsb.info.nih.gov/ij/download.html). To reduce variability in results, it’s suggested that multiple views of each well should be documented, and each experimental group should be repeated multiple times.


  1. Lampugnani M.G. (1999). Cell migration into a wounded area in vitro. Methods in Molecular Biology 96: 177-82.
  2. Yarrow J.C., Perlman Z.E., Westwood N.J., Mitchison T.J. (2004). A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods. BMC Biotechnol 4: 21.

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