Abstract
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
Equipment
Software
Procedure
Acknowledgments
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)].
References
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.
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).
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.
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.
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!
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.
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.
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.
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.
i wanna ask..after staining with crystal violet solution...is there need to remove the staining solution before taking photo?ASAP