Hi Tina, thanks for the reply.

I made a standard curve from 100uM, 50uM, 25 uM, 0uM nitrite, dissolved in the media that we treat our cells with (this media does contain and the standard curve changed colour in a dose dependent manner that was stable after a few minutes and did not change for hours afterwards. What was strange was that the samples did change (with more time and more reagent addition) while the standard curve did not. We thought that perhaps there was some other component in the media that was masking the colour change that was overcome by a longer incubation or secondary addition. What do you mean by the matrix interfering? We are considering using a more complex assay involving a reduction and/or deproteinisation step but are not sure what problem we are exactly trying to get around and therefore not sure what solution we need.

you should use DAN istead of griess which may be more sensitive. There are fluorescet kit sold by Cayman. After cytkines stimulation you should wait 4-6 hours sometimes 8 hours to see iNOS-derived nitrite production (you have to waiti that the protein is expressed). I would recommend 24 hours. Some cells (in particularly human cells) produce so low amount of NO that you cannot use Griess for measuements. Hope it helps.

Is the Griess reaction your only available option? It is not particularly sensitive way to measure NO.

You said that your standards contained 0-100uM nitrite - do you expect your NO being in this range? Even if inflammation is increasing NO greatly, it probably won't never come to even 1 uM, no matter what. In physiological conditions NO is in low nM range, so already 1uM would be 100-fold increase.

NO itself is almost never measured because it is unstable molecule and in low range. What people try to do measure is nitrite, the oxidation product of NO. Currently the most sensitive method to measure nitrite is chemiluminescence, which lets you to get down up to ~20nM-50nM range or so -with some luck and experience ;-).

As Barbora says the best way is chemiluminescence. I have one of this equipments in the lab and it has been quit reliable. If you need help let me know.

Hi Mark!

The Griess reagent is made to estimate nitrite. That means any NO developed has to be oxidized to nitrite. To ensure that this happens you can aerate the solution with air, careful not to loose any NO. However first you should be sure that the range and sensitivity of the assay (standard curve) is in the range you expected No is made.

Hello Mark! When you are trying to have reasonable results about NO production in samples using Griess reaction, you have to remember, that you detect only products of NO metabolism, and products could be different. For example, if cells are activated by inflammatory stimulus, they could produce more peroxynitrite than NO, and product of PN metabolism is mostly nitrate, as accepted, or nitrosated proteins et cet.

Also, as Barbora Piknova wrote, you can wait only micromolar NO concentrations from stimulated cells.

What you can do, it is to test limiting sensitivity of your kit: 10, 5, 1, 0.1 uM, and increase number of cells in detection condition.

Normally, the level of NO produced in inflammation is quite high, and if your kit is non toxic for cells and you can wait nitrite accumulation, it will not be a surprise for me, if you will be able to detect colour changes only after 1 hour following nitrite formation in the medium.

But to be more accurate, you could just change medium in your cells and then take probes for detection every 30 minutes, for example, to control nitrite accumulation.

Thanks for all the suggestions. I have been incubating my cells with cytokines (in particular, il-1beta) for 24 hours before collecting the supernatant. I will explore the possibility that the griess reagent is not sensitive enough and try a DAN based reaction, including a reduction step. I will keep chemiluminescence in mind as another alternative.

Thanks again, it's appreciated.

Rudolf, can I ask further about aeration. I wonder if this can explain the delayed results I was seeing (actually after leaving the solution out in the air overnight the colour of the samples changed profoundly, while the standard curve did not change at all) - is there are particular technique to aerate the samples?

Mark

The good news is that you only get the purple color from nitrite by diazotization, so if you are getting purple color it is coming from nitrite and not some unrelated compound.

Have you checked pH? The reaction needs acidic conditions and the symptoms you describe suggest your media may be buffering the acid and causing a slower reaction (fixed when you dump in more reagent).

As I know the incubation time for Nitrite with Griess reagent is at least 30 min not 15! And the ‎temperature is important too. For nitrite 25 C and Nitrate 37C, you should incubate 30 Min in these ‎temperatures to get good results

Suggest you read this http://www.promega.com/~/media/files/resources/protocols/technical%20bulletins/0/griess%20reagent%20system%20protocol.pdf?la=en

Didi you protect your reaction from light ?

they state in the doc: Incubate 5–10 minutes at room temperature, protected from light.

Hope it helps

Jacques

I would like to know here which Sigma NaNO2 you are using? cat no.237213 or some other? impurities in NaNO2 like chlorides and sulphates may also interfere in reaction i guess so that could be a reason why you are not getting increasing color change in your standard...

you could try DAFM instead

You can use NO specific probe- DAF-2DA for good result.

DAF does not react with NO but with one of its oxidation products, probably NO+. There is so far no specific probe which directly measures NO. One would have to use a Clark electrode or in order to be more sensitive, electrochemistry.

Developing a small colour during a long incubation means environmental nitrit "detection".