NOS activity in endothelial cells

Alison Leakey leakeyak at cf.ac.uk
Thu Jun 29 03:10:50 EST 2000


Hi everyone,

My problem relates to the production of NO and superoxides by
endothelial cells.
At present, I am culturing primary porcine aortic endothelial cells both
in traditional flat-bottom flasks (PRIMARIA) and on cytodex-3
microcarrier beads. Unfortunately, once I have obtained sufficient cells
to carry out experiments (usually passage 3) then there does not appear
to be any production of NO or superoxide. I use medium 199 with 20%
serum containing antibiotics (penstrep) and antifungals (amph B) and
passage using trypsin-EDTA, splitting 1:3.

Question: Will supplementation with L-arginine (NOS substrate) increase
activity?

Question: Will the presence of antibiotics cause a decrease in activity?

Question: Does anyone have any experience of how fast NOS activity is
lost from endothelial cells under culture conditions?

There have been many papers reporting that such activity exists in these
cells. WHAT AM I DOING WRONG?

Any suggestions would be greatly appreciated, Alison.
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