EC invasion into collagen gels and serum concentration - (Feb/28/2008 )
Has anyone seeded HUVECs on top of a collagen gel for an invasion assay? I have tried doing this and would like to add a growth factor to promote tube formation into the gel, once the cells are confluent. I've read that some protocols say to reduce serum (I use 10% right now) when adding VEGF to about 2%. Does anyone know why the serum is reduced? Should I do this?
Serum has thousands of things (e.g. growth factors) which might interfere with the VEGF effect. The serum concentration reduction is the way to ensure the cell response (tube formation) is due to VEGF (or any added vasculogenic factor) and not to other growth factor or molecule present in serum. I did some similar experiments but with endothelial cells differentiated from mouse ES cells seeded in ColIV. The serum concentration I used was 1% (the experiment was about 16 hours) but that was because differentitated ECs were too delicate to grow I'd try an experiment free of serum to check if other endothelial cell types can grow in that conditions... you know, to have a "clean" angiogenic response.
Well, hope it helps..... Good luck!
Thanks aleruiz! I will keep your comments in mind. One follow-up question, do you know of any reason why my advisor might be wanting the ECs to be subcultured in VEGF (~50ng/ml)? I thought that it was only to be used to activate the cells. He says that VEGF is a survival factor. I can't seem to get a clear answer.
Do you mean to grow ECs with VEGF 50ng/ml before the tube formation assay? Well, it's true that VEGF is a survival factor, but many other factors can perform a similar function. This subculture, is with or without serum? If cells won't have serum, maybe is a good idea to culture them in presence of a survival factor... although I'd rather prefer to culture them in presence of serum... Another possibility is that maybe your advisor wants to use cells "prepared" to tube formation in the way that VEGF can induce expression of its own receptor, so if ECs have plenty VEGF receptors, ECs will respond rapidly to the VEGF stimulation in the 3D culture to form micro vessels. If that subculture is short, let's say one day, I think it'd be the reason. Mmmm, I'll think about another possibilities and if another idea comes to my mind, I'll let you know.