I am using the scratch/wound healing assay as a platform to study cancer metastasis. This assay should mimic cancer cell migration for metastatic tumor initiation. However, after reading many manuscripts and protocols utilizing this assay, I found conflicting information on what percentage of serum to add to the cell culture media to allow for cell migration instead of proliferation. Many papers/protocols say to use normal growth media (10% serum for most cancer cells), but others say that if serum is present then what you will evaluate will be cell proliferation into the wound instead of migration. Some papers says use serum-free media and others have used as low as 0.5-1% serum. What is the correct amount of serum to use in order to mimic the human physiological environment to study cancer cell migration in this assay?
Serum starvation is an appropriate way to halt cell proliferation. So, starve the cells for at least 16 hours before you induce the scratch. You can also culture the cells in 0.5% serum containing media if at all you are worried about possible cell death by apoptosis or necrosis. This will still inhibit cell proliferate and you can be confident enough that the wound closure (if at all any) happened is only due to cell migration and not by cell proliferation.
Other way round (though you haven't asked about it), use Mitomicin C (2-10ug/ml depending on the cell line) to stop proliferation, 2-5h before scratching.
I have done this assay with the normal serum amount (10%) and I could not check any difference in the cell migration, comparing the treated to the control cells. I guess the cells should be FBS starved overnight before starting your assay, and it should not exceed more than 24h.
As a suggestion, there are some assays with confirm the cell migration more easily and reliably. Please, check search for migration assays using inserts. In this case, you can measure the cell migration through a membrane. Depending on the insert type, you can look at both physical migration and invasion, based on the collagen degradation.
Find the lowest level of FBS in which your cells survive and use this in scratch assay. As suggested by Vinod Nadella do a serum starvation for atleast a day.
Thank you Vinod Nadella ! I will try these suggestions. Thank you I will look into these options as well José Carlos Quilles Jr
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