I need to measure the level of nitric oxide in the cell compartment with a griess assay. The cell was exposed to the NOS gene and arginine, hoping to increase the level of nitric oxide. However, there was not much difference between sample and control group.
So.. what I wonder is...
1. Is that a possible to measure the level of Nitrite/nitrate with the cell supernatant? Is the NO derivative can be easily diffused through a cell membrane?
(I thought the cell membrane should be lysed to measure the NO related component.)
2. Are there any other main factors that influence the production of NO level except NOS and arginine?
Thank you for your help in advance
We always measure nitrite levels in cell supernatant (or in plazma) as follows: NO production is determined by measuring the levels of its oxidation end products NO2 and NO3. The data are then presented as nitrite levels of the total
amount of nitrite/nitrate after the conversion of nitrate
to nitrite by nitrate reductase in the presence of reduced
nicotinamide adenine dinucleotide phosphate (this step can be omited if the amount of formed nitrate is negligible). The nitrite
formed is then detected colorimetrically (540 nm) from
100 ul centrifuged supernatant by 100 ul Griess reagent
(1% sulfanilamide, 0.1% naphthylethylendiamine, and 2.5%
H3PO4) modified for 96-well plates. The nitrite levels are extrapolated from NaNO2
calibration curve. Good luck! With best regards. NKC
@Nikolina Kutinová-Canová
Thank you so much for your kind comment.
Can I ask you one more thing?
What's the exact meaning of cell supernatant?
I don't know which one of the below statement is correct
1. Cell membrane was lysed by lysis buffer and centrifuged to get a supernatant
2. Cell was cultured and attached in plate and the cultured media such as RPMI or DMEM is collected to be used as a supernatant sample. (Just take out the cultured media from the growing cell plate)
Your comment will be a big help for me.
Thank you.
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