Simultaneous detection of NOS-3 protein expression and nitric oxide production using a flow cytometer.
Nitric oxide (NO) is involved in the regulation of SMC proliferation during intimal hyperplasia as has been shown by the inhibitory effect on intimal hyperplasia of adenovirus-mediated ceNOS overexpression in injured arteries in pig. Good assays to quantify the NO-producing enzymes, i.e., NO synthases (NOS), are essential to analyze the mechanism of action of NO in this process. We have developed novel flow cytometric assays for the simultaneous detection of NOS-3 protein, using NOS-3 specific antibodies, and NO production using 4,5-diaminofluorescein-diacetate (DAF-2/DA). The presence of NOS-3 protein and NO production is demonstrated on human A549 and HepG2 cells infected with a NOS-3 adenovirus (Ad.NOS-3). A comparative study showed that the flow cytometric assays are equally sensitive as Western blot analysis, the citrulline assay, or the Sievers assay. On human endothelial and SMC, NOS-3 protein and NO production were simultaneously detected with the assays, both under basal conditions and after Ad.NOS-3transduction. Simultaneous analysis of NOS-3 protein and NO production, made possible by the here-described novel flow cytometric assays, is of significant value to those investigating NOS-3 and NO.
Crucell Holland BV, 2301CA, Leiden, The Netherlands.
M J
MJ
Havenga
B
B
van Dam
B S
BS
Groot
J M
JM
Grimbergen
D
D
Valerio
A
A
Bout
P H
PH
Quax
D004730:Endothelium, Vascular / Q000737:chemistry*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D009130:Muscle, Smooth / Q000737:chemistry*; D009569:Nitric Oxide / Q000032:analysis*; D019001:Nitric Oxide Synthase / Q000032:analysis*; D052250:Nitric Oxide Synthase Type III; D014407:Tumor Cells, Cultured
D009569:Nitric Oxide; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III