I tried two different kits to measure NO in HAEC cells; one from PROMEGA and another from ENZO lifesciences... although both work for the standard curve, the two kits failed to detect any signal in my samples.
I did the assay either with an endothelial cells specific medium (Promocell),or using DMEM without phenol red.. the result is always the same, nice standard curve but no signal in my samples.
Do you have any idea why it does not work? is it possible tha the concentrations of NO in my samples are too small? if so, how should I concentrate them?
Thank's for your answer Alain.
Indeed, that is what I did, but it doesn’t work. Concentrations are probably less than the kit detection threshold!
There are some excellent kits from Sigma, RnDSystems and Abcam, with relatively low levels of detection.
Good luck
You may not have much NO synthesis going on: it's not quantitatively high in endothelial cells and is dependent on/ promoted by lots of factors e.g. shear stress, hormones, cofactors etc that you probably don't have in a simple cell culture system.
If you would like to mesure in medium, you can use different kits for mesurement of secondary markers like cGMP (Cayman Comapany). But if you need real time NO levels, use DAF-FM. For me this is the best option
Hi Touat,
To b honest with you, Griess assay is very rough method, most of the times its nonspecific, which explains why you can't see any 'good' signal-to-noise ratio!!! Some good-standing journals don't accept the data either. If you don't have any other choice you could try the assay using positive and negative controls: L-NAME or something equivalent at higher concentrations and see L-NAME-inhibitable signal is different among different treatment groups! You haven't mentioned what was the treatment to induce NO generation in your assay, which also matters to some extent.
If you could afford a fluorescence assay using DAF-FM or cGMP, please respond, we can discuss about the experimental conditions.
Hi Touat!
First, you shoud define which NOS is your tartget. If you want to detect iNOS Griess may be OK but only with the controls that Yagna mentioned before. If your aim is to follow eNOS activity Griess is definitely not sensitive enough. (1 microM is the detection limit, however, eNOS produces NO in a 10-100 nM range). I tried several fluorescent methods as well (DAF-FM, DAF-FM-DA, DAR-1, DAN), electron spin resonance but none worked really well. E.g. DAF-FM-DA is a quite toxic agent at the concentration it should be used (10 microM), but has toxic effects even at 1 microM as well on HUVECs. The only method which worked for me more or less well is the ozone-chemiluminescence technology (e.g. GE Healthcare Sievers NOA 280i). This is specific and sensitive enough (1 nM), however, the cost of the instrument is horrible (~35.000 EURO).
PS: Whatever method you use, do not forget to supply your cells with 2 mM FRESH glutamine, otherwise they won't produce any NO...
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