HUVEC serum starving - (Nov/14/2009 )
I'm trying to use HUVEC for MAPK signaling assay.
Before I stimulate them with H2O2, I serum starved them in RPMI + 1% FBS for 16 hrs, but I'm losing large amount of cells.
I collected the lysis after stimulation and 2x wash with PBS, and I got around 50ug of protein per 90mm dish...
I was expecting a lot more...I think I lost too many cells during starvation and stimulation.
I heard HUVECs are hard to serum starve, I was wondering if anyone has any advice on this problem?
Thanks in advance for your help!
I've gotten my HUVECs from ATCC and I grow them in Vascular basal cell medium supplemented with the endothelial growth kit-BBE (bovine brain extract), in 2% FBS. I tried starving them with just the basal medium, no FBS, no kit but they basically die on my just after 4 hours. Even with FBS, they're not happy if they don't get the BBE kit.
WhenI starve them using 0.1% FBS, fully supplemented medium overnight, they look heathy and relatively fine the next day. Hope it helps.