For some reason I had to use HUVEC with high passage number (p8-p14).
I treat TNF-a (10ng/ml) for 1hr and administer my compound to see changes in the ROS level. In P5-P7 I got mostly 25% increase in ROS, but now my controls are giving more that that of TNF-a treated one.
Am I getting this because TNF-a action is somehow suppressed in those with higher passage number?
Another thing is EBM-2 + DCFDA (20uM) is giving a very very high RFU than that of others?
Since, I prepare TNF-a in EBM-2, so this is another very important issue I need to solve.
Thanks in advance.
While I can't answer your question specifically, the expression of all sorts of genes changes upon cell isolation and culture, and with increasing number of passages. As examples, BKCa and Nox1 are generally not present in native endothelium, but appear in culture. To give a general answer to your question, a trillion signalling pathways change when cells are cultured. It's a study limitation that all to often is not declared in papers.
Dear Deborah,
Thank you for your reply. I'll be much obliged if you could you share some of your articles.
Best