Hi,
I have a question, how do we determine the wound closure in scratch assay is due to cell migration and not cell proliferation? How can we rule out this? If I am not mistaken incubation period is very important, like we cannot incubate for longer. Usually, we just have to incubate up to 24 hours or less to prevent cell proliferation (mitosis)? Am I right?
Any comment?
Thanks
Seng
Well, in my case...I used serum-free media for 24 hours. Also you can let the cells starve for some time before the 24 hours begin. You can optimize your protocol trying various conditions....:) hope the attached file is helpful to you.
best regards,
Anna Van.
1. Check proliferation rate (if does not changes under your conditions then you are fine)
2. Use inhibitors of proliferation
3. Usually do not exceed 24 hours (till till then the proliferation rate is very low)
4. You are going to have always some proliferation, cell death etc, use appropriate controls always.
Dear Rajendra,
Yes, thank you for your additional information and reminder!
I have some experiences, i hope that helps
- keep cells at 95-100% confluence, and starve overnight (12-14h).
- or we can use Mitomicin C (2-10ug/ml) to stop proliferation from 2-5h before scratching. That depends on cell line.
- During scratching assay, FBS in medium should be 0.2-0.5%. That would help cells not going to apoptosis or necrosis, but also no proliferation occurs. Time for incubation could be 12h, 18h, 24h... Depending on cell line.
- Wound closure can be measured by ImageJ.
Dear Do Luong Huynh,
Another great and useful information given by you. Really appreciate!
Hi Yang-Seng,
Irrespective of which one(s) of the good suggestions above you will decide to go with (and especially if you wish to avoid using pharmacological inhibitors of mitosis), I'd encourage you to run another type of assay to be sure that your observations in the scratch assay are not limited to that particular assay, e.g. a short-term transwell migration assay (3-5 hrs) would be ok if done with all appropriate +/- controls; good luck!
If the time-lapse microscopy is availble (i.e. microscope with incubation chamber programmed to image every X minutes) - you should definitely use it instead of conventional time-point image capture
To avoid evaluating effects that would be secondary to changes in cell proliferation, in my opinion all recordings should be performed in presence of an inhibitor of DNA synthesis, such as Cytosine arabinoside (AraC; 10µM; Sigma).
See as an example our paper here attached.
Good luck!
I think you got all the answers you need and you answered yourself, to use mitosis inhibitor such as mitomycin C. I am not a big fan of wound healing due to the variation of size of wounds. If you want to go something fancy, this may help http://www.emdmillipore.com/US/en/product/Cell-Comb%E2%84%A2-Scratch-Assay,MM_NF-17-10191
Like Vladimir said, I would recommend to use transwell migration with cell culture inserts (and invasion with any ECM matrix coating). It is quantitative with less deviation, without concern of cell proliferation although you have to buy the inserts.
I've done this assay many times and the trick is to serum starve the cells for at least 16 hours before you produce the scratch wound. This will inhibit the cells' ability to proliferate so you can be sure that wound closure is due only to cell migration.
I agree with Lenora Pluchino that serum starvation is appropriate way to halt proliferation. We have done wound healing assays using serum starved cells (16-18h) followed by wounding, washing and incubation with serum free medium.
Much appreciated. I am trying to do this experiment now. Your suggestions are really very helpful.
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