we are using griess reagent (0.1% naphthylethylenediamine dihydrochloride, and 1% sulphanilamide in 5% phosphoric acid) for estimation of NO.
few months before we were getting very good absorbance at 540 nm and recently when we did the test the absorbance was very less..both with our test samples and standard sodium nitrite also...
what may the reason?
can we use colored media like DMEM when estimating NO in cell culture supernatants?
Well this can happen when the solutions for Griess are not made freshly, or not mixed well, but there is nothing else that i can think of...
As for the medium (DMEM) I think its better to avoid colored medium, at least that is what we do. But if you can try measuring the absorbance at 540nm of pure medium and see what happens.
All best
Agree with Nikola on reason for low colour.
With regard to media containg colour indicator: We do Griess assays routinely in coloured DMEM. It works fine. Do not use RPMI as it gives very high background readings.
Prepare fresh HBSS from powder with 18 mOhm DDH2O and add NaHCO3 to wah the cells. Then add the HBSS onto cells for the experiments (may add arginine 20-25 nM to increase the difference). One problem we had was the nitrite/nitrate already in the culture medium which may give us problem due to strong background signals. If the background signal is several fold higher than what the cells can generate then your results will be chaotic after correcting against the blank. Our cells were pulmonary artery endothelial cells which may be different from your cells.
As has been mentioned, make sure you're preparing your reagents fresh.
For what it's worth, I always added the reagents serially, so Sulf for 5-10min, then the NED.
Also, any chance of some changes in your water or other reagents? or perhaps spectrophotometer settings?
Regarding DMEM or other media, phenol red shouldn't affect it much, it will be yellow due to the acid anyways. I'd be more worried about high protein systems. Regardless, you simply need to make sure you prepare your standards in the same matrix. Also, some spike recovery tests are always a good idea.
I have to add one thing: you can not directly measure NO by the Griess method. It is more or less measuring nitrite/nitrate in your cells/medium. The value you receive from your measurements can only give a hint of NO production.
Perhaps you should switch the assay if you really want to measure NO concentrations.
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