I have performed few of the initial experiments for HUVEC cell migration in BD 24 well FluorBlock fibronectin coated plates. I procured the pre-screened primary HUVEC cells from Promocell. I am able to get 8-10 fold migratory response with 5%FBS after 24hrs but only around 2 fold migration with 5ng/mL VEGF when detected with Flexstation-III after Calcein staining. Tried overnight fasting of cells before the assay. Also increase in VEGF concentration didn't increase the migration. Please suggest how can I get better response?
when you starve the cells overnight, wash them first with PBS 2-3 time to remove any residual FBS. Also you might want to try different cell lots since the expression of VEGFr is variable in different HUVEC lots. Is your VEGF fresh and not subjected to more than one freeze thaw?
Hi Ankur,
No single chemoattractant will be as potent as FCS in attracting cells. The overall migratory capacity of cells is higher in FCS as it they can draw energy from it more easily. That being said, 5ng/ml is pretty low for VEGF.
I did a dose response curve for migration of endothelial cells towards VEGF in a paper a while back (1), I have attached the graph here. The EC50 is around 20 ng/ml the optimum at around 50ng/ml. Go higher and your gradient becomes unstable.
At any rate your response seems pretty normal, 2-fold isn't so bad.
1. Poldervaart MT, Gremmels H, van Deventer K, et al. Prolonged presence of VEGF promotes vascularization in 3D bioprinted scaffolds with defined architecture. Journal of Controlled Release. 2014.
Thanks all for you suggestion.
Thomas, I tries at 100K/well density that was showing lesser response as compared to 50K/well
Hi George,
Right now I have only one lot of HUVEC cell that I purchased from Promocell. These are prescreened cells. VEGF was freshly prepared in 1xPBS with 0.1% BSA. Ihave also made the aliquotes of it just to avoid freeze thaw cycle.
Hi Hendrik,
Thanks a lot for your input. Recently we tried till 20ng/ml conc. of VEGF. Could not get more than 2.2 fold migration. I don't have access to your article. Can you please share?
Hi Ankur, I've made the article available via researchgate, you should be able to access it in the link below. If you look at fig 3a, which is modified Boyden chamber migration, you'll see we didn't see that much of an effect either, around 2-fold at best.
The dose response above is with the real-time impedance system, which is a bit more accurate, still, effects aren't huge either. You do horizontal migration with monolayer defects, right?
Regarding your VEGF, it's the same preparation/storage method we used, although for longer term storage I recommend -80 C. There may also be brand differences in VEGF potency, we have good experiences with R&D.
- Hendrik
Article Prolonged presence of VEGF promotes vascularization in 3D bi...
Thanks a lot Hendrik. I use Boyden chamber for transwell migration assay. These chambers are precoated with fibronectin purchased from Corning/BD. Well, now I think 2 - 2.5 fold is a good enough challenging with my test compound. Hopefully I should get some significant data.
Thanks everyone for your valuable inputs.
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