I mean 30ng per well. I am trying to elicit a VEGF stimulated calcium response from them, by assing 30ng to a well of confluent cells loaded with Fura2AM. I have tried this in mouse liver endothelial cells extracted with CD146 beads and my colleague pulmonary ECs with CD31 beads.

It has just occurred to me we are using human recombinant VEGF. I gather there is 80% homology of the mouse and liver versions but I am going to try the mouse recombinant also.

I wondered if anyone had seen a calcium response from them using VEGF (we are a calcium channel lab...)

You said that the culture medium already contained VEGF. What is the concentration? If the medium has a level high enough of VEGF, an extra add of VEGF might not give a response.

Hi

Maybe you should starve your cells using non-supplemented EBM2 media, this will synchronize your cells and allow for a better response. Also you mentioned the use of confluent  layers, this is not the best setting to check for a VEGF mediated response in endothelial cell lines. Maybe a 75% confluence is a best choice. Also I suggest you  to check for a proliferation response in accordance with the IC50 of the VEGF you are using.  There is no difference between human or murine VEGF  effects on endothelial cells, the site for binding VEGFR2 is almost identical.

good luck!!

As said above, the cells' proliferative response may be max'ed out by the media.  You should switch to serum - free or low serum. Also 30 ng/well is quite high and probably translates to 120 ng/ml, which is pharmacologic. I would try a dose curve of 1-10/ngml 

I apologise I dont think I have explained the experiment very well. I am testing for calcium release stimulated by VEGF. This requires a confluent monolayer of cells for the machine to detect changes in intracellular calcium in cells that I preload for an hour with Fura2AM in SBS before putting them into plain SBS. The machine adds back 30ng VEGF to the wells and it detects changes in intracellular calcium, which it wont detect reliably with subconfluent cells. We do this experiment in HUVECs routinely and get a response.

Up until that point the cells are cultured in EBM2 media at around 5nM VEGF, as provided within a commercial media supplement kit.

I have stained for VEGFR2 and they are present. I just cannot get this response. I have not tested their proliferation with and without VEGF though or any other functional experiments such as migration assays, as my supervisor was keen to determine a VEGF response first.

What sort of experiments would you starve them of VEGF and serum ahead of? Would you do this for testing for calcium response? If so how long would you starve them for?  I have not heard of this before except for proliferation assays and cell cycle analysis.

Thanks for all your input, any advice is greatly appreciated

You may also try both normoxic and hypoxic conditions.  In my study of cancer cells, the VEGF pathway is augmented by hypoxia.

Ah I have read a bit about papers serum starving before testing for VEGF responses now, now I understand!! Many thanks for your wonderful suggestions. I shall try the serum starving and dose response and let you know

CD146 is also expressed by fibroblasts, how do you seperate the contaminated fibroblasts from your mouse liver endothelial cells?

I apologize if this is simplistic, but unusually elicit a strong VEGf response by placing the cells into a hypoxic chamber of incubator.

I tried a second pass but haven't been able to, fibroblast growth was a big issue,  so I have abandoned this protocol.I was going to try CD31 and do a second incubation with CD105