I am trying to measure the Nitric oxide production by RAW 264.7 macrophage on infection with influenza virus using NO assay. I infect the cells for 5 hrs and measure NO at 12hrs and 24hrs. Standard used for comparison is Sodium Nitrite. The NO production by RAW 264.7 infected with influenza virus is similar to the NO produced by Mock cells. However, when I carry out phagocytosis assay, I get a good amount of percentage of phagocytosis (same condition used as NO assay).
I would like to know if there is any co-relation between NO production and phagocytosis and also is there any alternate method / solution for NO measurement.
There indeed is an established relation between phagocytosis by macrophages and nitric oxide (NO) however people are still working on the precise role of NO in innate immunity. Upon phagocytosis, macrophages (RAW 264.7 is a murine macrophage cell) produce a variety of active cytocidal (germicidal) agents that help kill the engulfed microorganism.They may be ROS like hydrogen peroxide (H2O2) or superoxide anion (O2–) [produced by lysosomal NADPH oxidases] that directly oxidize pathogen macromolecules as well as nitric oxide (NO),produced by the inducible isoform of nitric oxide synthase (iNOS) which react with superoxide to produce the potent RNS, peroxynitrite which causes extensive nitration of proteins and DNA of the engulfed pathogen leading to their destruction. Macrophages may also deploy other agents like lysozymes, defensins and lactoferin for accomplishing such pathogen destruction.
It is not clear what method you have used for your NO Assay. Most probably you have used the Griess Assay as you indicate that you have used a sodium nitrite standard curve. Interestingly assay of nitrite produced in the cell media does not give you a precise idea of the source of NO generated in a physiological setting as NO is not only produced by NOS. You may conduct a oxyhemoglobin assay using fresh RAW cell extract to measure the NO produced specifically by iNOS.
I have no idea about iNOS expression in RAW 264.7, but working with avian peritoneal macrophages, I found more or less similar trend in phagocytosis and NO production against SRBC (as particulate antigen) LPS challenge, respectively. The can be found in attached article.
Dear Keerthana Sekar?
You wrote:
"I would like to know if there is any co-relation between NO production and phagocytosis and also is there any alternate method / solution for NO measurement".
I will answer the first part of the question. Yes, there is co-relation between NO production of macrophages and phagocytosis.
There is co-relation between NO production of macrophages and NBT-test.
Thank you for all your kind reply.
Koustubh Panda: Yes, I use Griess Assay. I get a good result when I carry out phagocytosis assay but under same conditions when I carry out NO assay, after infecting the cells with virus, the NO measure looks similar to mock.
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