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Scratch Wound Healing Assay    

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The scratch wound healing assay has been widely adapted and modified to study the effects of a variety of experimental conditions, for instance, gene knockdown or chemical exposure, on mammalian cell migration and proliferation. In a typical scratch wound healing assay, a “wound gap” in a cell monolayer is created by scratching, and the “healing” of this gap by cell migration and growth towards the center of the gap is monitored and often quantitated. Factors that alter the motility and/or growth of the cells can lead to increased or decreased rate of “healing” of the gap (Lampugnani, 1999). This assay is simple, inexpensive, and experimental conditions can be easily adjusted for different purposes. The assay can also be used for a high-throughput screen platform if an automated system is used (Yarrow and Perlman, 2004).

Materials and Reagents

  1. Human MDA-MB-231 cell line (ATCC, catalog number: HTB-26 ™)
  2. Dulbecco's modified eagle medium (DMEM) (Life Technologies, Invitrogen™, catalog number: 10313-021 )
  3. Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
  4. Phosphate buffered saline (PBS) (Life Technologies, Invitrogen™, catalog number: 14190-144 )
  5. Glutaraldehyde (Sigma-Aldrich, catalog number: G6257 )
  6. Ethanol (Sigma-Aldrich, catalog number: 459836 )
  7. Crystal violet (Sigma-Aldrich, catalog number: C3886 )


  1. BD Falcon 24-well tissue culture plate (Fisher Scientific, catalog number: 08-772-1H; BD Biosciences, catalog number: 353226 )
  2. Rainin pipet tips (1 ml) (Mettler-Toledo, catalog number: GPS-L1000 )
  3. Cell culture incubator: 37 °C and 5% CO2


  1. Photoshop or ImageJ (


  1. Grow cells in DMEM supplemented with 10% FBS.
  2. Seed cells into 24-well tissue culture plate at a density that after 24 h 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 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.
    Note: Medium may contain ingredients of interest that you want to test, e.g., chemicals that inhibit/promote cell motility and/or proliferation.
  7. Grow cells for additional 48 h (or the time required if different cells are used).
  8. Wash the cells twice with 1x PBS, then fix the cells with 3.7% paraformaldehye for 30 min.
  9. Stain the fixed cells with 1% crystal violet in 2% ethanol for 30 min.
  10. Take photos for the stained monolayer on a microscope. Set the same configurations of the microscope when taking pictures for different views of the stained monolayer. The gap distance can be quantitatively evaluated using software such as Photoshop or ImageJ ( 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.


This protocol was developed in the Department of Immunology, Scripps Research Institute, La Jolla, CA, USA and adapted from Lampugnani (1999) and Yarrow et al. (2004). The work was funded by NIH grants CA079871 and CA114059, and Tobacco-Related Disease, Research Program of the University of California, 15RT-0104 to Dr. Jiing-Dwan Lee  [see Chen et al. (2009)].


  1. Chen, Y., Lu, B., Yang, Q., Fearns, C., Yates, J. R., 3rd and Lee, J. D. (2009). Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration. Cancer Res 69(8): 3713-3720.
  2. Lampugnani, M. G. (1999). Cell migration into a wounded area in vitro. Methods in Mol Biol 96: 177-182.
  3. 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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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Chen, Y. (2012). Scratch Wound Healing Assay. Bio-protocol 2(5): e100. DOI: 10.21769/BioProtoc.100.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

Carl Lee
internal medicine of University of Utah
Is the cell in growth status or starvation status, means the concentration of FBS should be 10% or 0.1%-1%, when the cell treated with drugs? Thank you.
12/1/2014 2:13:09 PM Reply
Yanling Chen
The Scripps Research Institute

The protocol described here is for what you called "growth status", and you can modify it according your experimental requirements-it really depends on your assay goals and cell type. As you probably know, many cells (even cancer cells) in low serum media won't grow as well as in high (i.e., 10%) serum conditions. If your purpose is to maximize the effect of a drug under low serum conditions, you can try your cells of interest at lower FBS for sure, but the assay conditions should be empirically determined (for a better "combination" of assay settings).

12/2/2014 7:31:42 AM Reply

larita salv
I'm having some troubles reproducing some knockdown results.
Sometimes, independently of scratch width, my scratched cells migrate very poorly and after more than 48h wound is still open and cells alive.
Does it ever happen to you? Any advice?
Another question: does sodium pyruvate in the medium affect wound healing?
Thanks a lot!
8/4/2013 10:06:01 PM Reply
Yanling Chen
The Scripps Research Institute

Outcome of the "healing" process very much depends on the cell type, i.e., those that migrate aggresively/spread out quickly probably will heal the gap much faster, such as the example cell line mentioned in the protocol. Also, the "healing" process is resulted from not only cell migrating, but also cell growth. finally, please select enough views and repeat the same assay several times-just to reduce variability in readouts so that they are objective and reliable (statistically).
And I don't think sodium pyruvate would have negative effect on would healing.
Hope that these could help.

8/6/2013 7:20:19 PM Reply

Lisha Chen
Hi, I would like to know whether I should coat some ECM like Matrigel or other proteins? Some articles I have read recommed protein coating before experiment. Thank you!
5/28/2013 8:38:13 PM Reply
Yanling Chen
The Scripps Research Institute

You may coat the surface with some proteins as long as the protein layer is even and uniform. For Matrigel, I think it's more difficult to scratch the surface to generate a gap, and the cells may grow into the EMC and stay there (not grow to "health" the "gap"). Please try and find a better and creative way for your experiments.

5/29/2013 7:18:24 AM Reply

Lisha Chen

Thank you! I have another question. Should I change the medium to the serum free medium before I scratch the line? Since I need to detect a cytokine activity. When I detect the proliferation activation, I always change to a serum free medium to starve the cell before add the cytokine. Thank you!

5/31/2013 12:09:35 AM Reply

Yanling Chen
The Scripps Research Institute

I think switching to serum-free medium should not be a problem as long as your cells can survive and grow well (for some time) in the serum-free medium. But please keep in mind that some cell lines need serum to maintain normal metabolism and other functions such as spreading out or moving etc.

6/4/2013 6:20:12 AM Reply

rose luo
is this assay can evaluate the nitric oxide-induced endothelial cells dysfunction?Thank you a lots!
5/6/2013 11:56:15 PM Reply
Yanling Chen
The Scripps Research Institute

This assay is used to study the outcomes of a specific treatment of cells for cell migrating and growth. I don't have laboratory experience with assays of nitric oxide induction of endothetial cells. If you are to test cell migration and growth as an experimental endpoint, you may want to modity the assay design for that purpose.

5/9/2013 2:14:25 PM Reply

Carmen Toro
I would like to make this assay (modifying something) and then trypsinize the cells in order to obtain single cells to do patch clamp on those cells. Is that possible? How can I to obtain two groups: migrating and no migrating cells? Could you please give me some suggestion?
Thank you in advance.
5/8/2012 11:36:22 AM Reply
Yanling Chen
The Scripps Research Institute

For the patch clamp assay, you probably have to try it because I do not have this information.
For seperating/obtaining migrating and non-migrating cells (again sorry I don't have this info). You might want to try a different method, e.g. cell invasion assay using matrigel and then retrieve the migrated cells from the gel? Just my suggestion.

6/1/2012 1:34:55 PM Reply

why there is need to stain with crystal violet? can we visualize the cells inside the wells or we have to take out the round coverslip?
3/25/2012 9:01:52 PM Reply
Yanling Chen
The Scripps Research Institute

Staining with crystal violet (other other dyes of your choice) would make visualization easier; also better for taking pictures.
And if you need to grow cells on coverslips (that may be coated with reagents of your interest), you will need to modify/optimize the protocols.

6/1/2012 1:34:37 PM Reply

nor hidayah

i wanna ask..after staining with crystal violet there need to remove the staining solution before taking photo?ASAP

4/3/2014 5:05:37 AM Reply

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