With Griess reaction you can measure only nitrite, if you have no an additional reaction to detect nitrate after reduction to nitrite, that is included in procedure of some kits. Nitric oxide could be converted to both compounds in culture cells depending on redox condition, oxygen level/consumption, another targets et cet.

You have to check also the calibration curve, if you have got the limitation that was promised by the kit supplemental in your hands. And you can decrease, for example, a ratio (medium : cells), to have higher concentration of nitric oxide secondary products in the medium.

Normally, culture cells produce very high level of nitric oxide after LPS administration (about 24 h.) as we detected by the EPR spin trapping.

hm. hello. i used to detect NO in TC medium as converted nitrite due to its spontaneous oxidation. i used home made Griess reagent. I had never problem to detect the secreted NO/nitrite in complete TC medium, however, i worked on macrophages and glial cells, they secret a hugh amount NO during LPS stimulation.

if iNOS activated in your system, you should detect some increase after 24 hours incubation/exposition at least.

Shortly, my answer is your last two question is yes.

To get iNOS transcription, you need the full medium during stimulation. (I do not know how you measure your NO, so for detection, i have no clue.)

I done some studies on measuring NO increase in skin fibroblasts stimulated with H2O2 or TNF-alpha and I could see differences. The mesurements where done in complete medium and I did these tests using two different kits which are based also on two different methods: one was using chemiluminescence variations in order to see the peroxynitrite elevation - see this link:

http://www.knightscientific.com/test-kits.html

The other one was from based on Griess reagent and it was QuantiChrom™ Nitric Oxide Assay Kit from BioAssay Systems.

I can tell you that the kit from Knight Scientific it was more sensitive but it is also more time consuming. Good luck with your work, is a very interesting parameter to measure and you could make very interesting connections with other cell mechanisms after you measure this one.

With Griess reaction you can measure only nitrite, if you have no an additional reaction to detect nitrate after reduction to nitrite, that is included in procedure of some kits. Nitric oxide could be converted to both compounds in culture cells depending on redox condition, oxygen level/consumption, another targets et cet.

You have to check also the calibration curve, if you have got the limitation that was promised by the kit supplemental in your hands. And you can decrease, for example, a ratio (medium : cells), to have higher concentration of nitric oxide secondary products in the medium.

Normally, culture cells produce very high level of nitric oxide after LPS administration (about 24 h.) as we detected by the EPR spin trapping.

To add to this there is certain bio-sensor method, which can detect minute redox change in the system so in cell culture derived NO may be detected by such method after proper standardization.

What method have you used to measure Nox levels?

I have examined NO production and iNOS expression in both Rat Smooth Muscle Cells (RASMCs) and J774 macrophages. Cells in culture are normally serum starved in order to eliminate proteins contained in the FBS from deactivating normal enzyme activity (example as seen in adherent cells when serum based media is used in washing cells to deactivate the enzyme trypsin after a trypsinization procedure). Hence the use of inhibitors further downstream will enable one verify that it is really the inhibitor and not the serum proteins. I suggest after a 24hr serum starvation, you then proceed to culture in full (10% FBS) media for a further 24hr before stimulating the cells for NO production (at least an18 hr incubation period in presence of stimulant(s)).

Secondly in cell culture, it is always best to start your actual ‘test’ experiments with passage 3 – 5 when the receptors on cell surface are at their maximum and/ or fully mature. I am not conversant with optimum level of concentration required for inducing iNOS expression in this particular cell type but a dose response of both stimuli might be helpful. Also see reference by Catarzi, S et al. Inflamm Bowel Dis. (2011) in which LPS (1–10 µg/mL) was used.

For NO detection I have always used 100 µl of homemade Greiss reagents with 100 µl of cell media (supernatant) mix without any problems. Hope this works for you!!!

Dear all,

Thank you very much for your responses.

Carmen Vazquez, the method I used was griess (Sigma kit). This method allows me to measure nitrites and nitrates in the culture medium. The kit includes nitrate reductase and co-factors to convert all the nitrate content into nitrite.

All the best.

Hi Juan,

Were you able to observe Nitric Oxide increases using the kit with nitrate reductase. I am also facing same problem where there is no difference between control and the treated one. Even when I used DMEM F-12 without phenol red.. the blank (media without cells) gave higher reading than control and treated one with no change between the latter two.

Any suggestions??

Hi Apurva,

Unfourtunately, I could not see any effect of IL-1b or LPS on NO production. I think that the level of NO in culture medium was under the limit of detection of the method. According to Paduch and Kandefer-Szerszeń (2011) (article attached), the levels of NO produced by myofibroblasts of colon CCD-18Co (the cells I used) were below 1uM, whereas the limit of detection of the method I used was 10 uM.

Try to find some publications indicating the basal level of NO produced by your cells. Perhaps your should use a more sensitive method to detect it.

Another possibility could be that your treatment does not induce NO production in your cells. Have you found previous publications describing induction of NO by your treatment?

Should you have any question, please let me know.

Best,

Juan.

Hi Irina,

NO was estimated in our laboratory from a treatment media that is composed of Senna leaf extracts (crude). I incorporated Griess. Previously, upon adding the solution to the medium, a pink colour developed which indicated release of NO in the medium. We use the plant extracts to treat Hymenolepis diminuta (cestode parasite). But now the same experiment has been repeated, but no results are coming. We dissolve the extracts in phosphate buffer saline and add 1% dimethylsulfoxide for increasing permeability of the extracts to the parasites. After paralysis, we took the medium, centrifuged it and added Griess solution to it, but no development of colour occurred. Could you kindly give some clues to why this thing is happenning? If the parasites are getting paralysed then stress must have occurred and there must be a release of NO in the media?

Hi Bidisha,

If I have understood correctly, you cannot have the same outcome from the Griess test that you have in previous experiment. You can compare your protocol with previously published, for example: https://www.promega.com/~/media/Files/Resources/Protocols/Technical%20Bulletins/0/Griess%20Reagent%20System%20Protocol.pdf

Then you can take into account storage conditions for the reagents, sensitivity to nitrite of Griess reagents in appropriate medium and prepare calibration curve (with nitrite); next, analyze if the quality of the plant extract and biological materials was not changed, as well as time of incubation of plant extract with H. diminuta, concentrations etc. Finally before definite conclusions, you have to take into account negative controls: test medium form the same amount of plant extract, and medium from the parasites treated with vehicle only (w/o extract).

The reaction of the parasites to stress generally could involve different mechanisms I suppose