I would like to measure the change in nitric oxide (NO) that cultured cells produce in response to ultraviolet light. NO appears to be difficult to measure because of its short half-life. Is it best to measure NO released into the cell culture media or intracellular NO. What is the best method to use for both of these. Any thoughts and suggested appreciated.
Thanks, Ken
Freeze the culture immediate at -70oC. Using nitric oxide kit you can detect the nitric oxide in the media.
You cannot measure NO by the Griess Reaction (a commercially available assay kit). The Griess Assay will only measure Nitrite in the culture media. It will not measure the Nitrate in the media.
NO degrades to both Nitrite and Nitrite....so to measure the total you need to use Griess AND a Nitrtae Reductase assay
As NO degrades to both Nitrite and Nitrite, it can't be measured directly for this you have measure nitrite and nitrate seprately......
To measure the nitrite, you can use Griess Method
Further for nitrate determination, nitrate reductase assays are there
in my opinion, after treating the cells with the factor of interest, proceed with cell harvest and collect both pellet and supernatant. The supernatant will be tested for NO assay while the pellet will be used in iNOS expression via Western Blot. Then, maybe you can correlate both results. Correct me if I'm wrong. Thanks =)
Dear Ken Raj, I know very good NO specialist. His name is Ernst van Faassen. He has a very nice book "Radicals for Life: The Various Forms of Nitric Oxide. ISBN13: 9780444522368" about NO detection (possibilities, problems, etc.). The level of NO production by different cells is different and not every method is sensitive enough to detect NO. We used the EPR (Electron paramagnetic resonance) technique to detect low NO level in the suspension of bone marrow cells (http://www.ncbi.nlm.nih.gov/pubmed/23554866). With EPR techique and knowledge of Ernst van Faassen we were able to detect the basal NO level in the cultured HUVEC, brown fat cells, etc. I guess that if you will contact Ernst van Faassen ([email protected]) you will gat a grate answer on your question, with all possible detailes and pitfalls, suggestions, etc.). Just look at his articles in Pubmed and you will see that he is the right person to contact with. Success!
Dear Ken,
this paper could be helpful for you. A novel class of genetically encoded NO probes for live cell imaging.
best wishes
http://www.nature.com/ncomms/2016/160204/ncomms10623/full/ncomms10623.html
We used the EPR (Electron paramagnetic resonance) technique to detect low NO level. Nitric oxide is bound by diethyl dithiocarbamate and iron introduced into the medium
Gopinath V.P hello, can you describe after >>>Freeze the culture immediate at -70oC. What 's the next step?
Thank you
Best regards,
Andy Wijaya
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