Does this mean that no one knows how to do it?

I would choose another cell line (e.g.: BAEC, BLMVEC, human microvascular endothelial cells). HUVECs don't produce much NO and usually people preferer using another NO sensor such as cGMP in HUVECs

HUVEC do not produce much basal NO.  I do not know what conditions will be you using.  One of the problems of using nitrite/nitrate is that you have to control for the culture media.  Depending on the batch serum can have large amount of nitrite/nitrate that mask changes in HUVEC cell production.  I think you should use a different method to measure NO production if you do not want or cannot change the cell model.  Does not matter what method you use, you will need controls, lots of controls

Thank you for your attention and participation. Unfortunately, we have to use HUVECs for this assay as we want to prove that results published recently are not exactly true and it looks that the authors of the paper very loosely interpreted the results (published in a very good journal). Protocol is poorly described in the M&M section, only general information about what kind of assay was used to determine Nitric Oxide production. There was almost nothing about the cell culture.

Thank you again!

I would recommend fluorimetric detection of NO. The amount of NO produced depends on the type of stimulation. If it is acute response like estrogen, insulin, catechin, you should not stimulate more than 45 min. It is very critical to standardise the time point of your active. Griess is relatively less sensitive. 

I would recommend 24 well plate, with 250ul volume of stimulation.Serum starvation increases your agonist induced response, but it may also increase your basal NO levels. Ideally 4-6 hr of starvation is optimum. do not starve in low glucose medium.

Stimulation has to be given in Hanks buffer, where nitrate levels will be minimum. do not you medium, as it contains nitrites/proteins.

Please perform your experiment same day, do not store. 

Thanks Paul!  You've just made my day! 

Welcome. Another important point is to use. basal media for starvation (without endothleial growth factors or any other growth factors). Hope this helps. Please let me know for any more queries.

Best wishes

Dear Marcin Chrusciel, I would recommend you to contact to an expert in the NO field (Dr. Ernst E.H. van Faassen, [email protected]). He knows a lot about the NO detection. We were also able to detect the basal NO production by the HUVECs (page 6 in this article http://www.ncbi.nlm.nih.gov/pubmed/23554866 ). Success!

Thank you Marina.

Dear Marcin Chrusciel,

I am facing problems to detect nitrite and nitrate in HUVECs medium. As you raised this question 5 years ago, I wonder if you succeeded?

I use HUVEC in passage 2-5, I starve them for 2-3 hrs before the stimulation. The stimulation is added in complete medium (LONZA) for 2-3 hrs (according to the manufacturer there is no nitrite or nitrate in the medium and the phenol red concentration is very low). I am using fluorescent method based on DAN reaction (from CAYMAN chemicals).

I can not get results that repeat themselves, even with the positive control (I tried acethylcholine and LPS).

I would appreciate any help regarding the assay.

The only reliable methods for measuring NO in HUVEC are reductive chemiluminescence and EPR. Giess assay is not sensitive, and the medium per se have a large range of nitrite and nitrate, making any NO coming from the cells disappear in the basal medium from cells. DAN does not measure NO.