I am currently performing scratch wound healing assay using HUVECs. I serum-starved my cells overnight prior to wounding my cells. I used EBM + all supplements except growth factors + 0.1%FBS as my starving media, as I found that the cells were not healthy (they start to lift and die) if I only use EBM without any supplement. I had BBE in my starve media.
Interestingly, 24 hours after human VEGFA stimulation, I did not manage to see an effect in the migration area of VEGF group compare to no VEGF control group. This is quite surprising considering VEGF is a potent angiogenic factor used in many HUVEC migrations studies (I used 25ng/ml, same as many other studies). Our VEGF and HUVEC batches are new and we used P3, cells looked healthy after 24 hours migration so the issue is unlikely to do with the cells. Could it be the starving condition? Can someone who has done this offer a suggestion?
Hello Vincent,
If you used the supplements in normal concentrations, using the supplements in lower optimized concentrations might help to see the effects of VEGF.
For HUVECs it is possible to see small differences instead of observing a huge difference. VEGFr expression is variable for different HUVEC batches so, it might be helpful to use a pool of different cell batches to see the effects.
For the lifting issue other than the supplements, it might be also helpful to check the coating method.
Also, you can try increasing the serum to 0.5% and maybe decrease the starvation time for observation to keep the cells more healthy.
Good luck with your experiments.
I once dealt with a batch of endothelial cells that would not respond to VEGF in a migration assay. After endless testing and trying, for which I wasted a lot of time I binned the cells and ordered a new batch.
If you have the option to do a transwell chemotaxis assay, which is in many ways easier that a scratch assay, then try that.
Mustafa Beter Anette Magnussen
Thanks a lot for your suggestions! I am planning to run an optimization experiment with different concentrations of FBS and supplements. Will keep you updated with how it goes.
In my opinion, this sounds like a problem in the experimental setting itself. Normally, HUVEC are responsive to VEGFA.
I would propose to use a novel set of cells (optimally primary isolated or a new ordered batch, maybe of a new purchaser.
Another possible source is the VEGFA itself. Possibly it is not active or not potent enough compared to other samples/batches.
These two sources should be checked or maybe corrected to make HUVEC moving following the VEGFA gradient.
Hi All,
I just realised that I have completely forgotten to reply for almost a year! I did manage to optimize the protocol, and in the end my HUVEC got a pretty good response to VEGF (more than 150% migration 18 hours after treatment). I think the trick here is not to add BBE or any other supplements into the EBM starve media, other than FBS (0.5%), Glutamine and ascorbic acid. It is possible that these other supplements/growth factors may have other effects on the HUVECs (e.g proliferate) rather than migrate, which may be why I didn't see a response to VEGF. Also I did starvation for 4-6 hours only and not overnight.
Thanks for all your contributions! If you have an even better protocol, please do share.