I have estimated nitrite content in tissue homogenates, but anyhow the procedure remains the same for all.

Nitrite is estimated following "Griess Reaction", which is based on two-step diazotization reaction in which acidified NO2- produces a nitrosating agent, which reacts with sulfanilic acid to produce the diazonium ion. This ion is then coupled to N-(1-naphthyl) ethylenediamine [NEDD] to form the chromophoric azo-derivative which absorbs light at 540 nm.

Procedure:

-Equal volumes of sample (tissue homogenate/Media) and Griess reagent (5% phosphoric acid containing 0.1% NEDD, 1% sulfanilamide) should be mixed and incubated in dark for 10 min at room temperature.

-Then the absorbance of the reaction mixture is measured at 540 nm.

-The concentration of nitrite in the sample can be determined from a sodium nitrite (NaNO2) standard curve. (for this you can first check the absorbance of one of the samples, which you feel will give the highest concentration and then based on that you can plot the CC curve)

Even-though the end product is violet colored, I feel that using colorless media is better,since any color i your samples may give you erroneous absorbance values..

Dear Pandian Nagakannan,

Thank you so much.

What is the highest concentration of sodium nitrite used in the standard curve?

Hi Jessica,

The range of standard curve can vary according to your requirement/s. Usually I use 1.56µM to 100 µM and for some assays, 1.56µM to 10 µM.

Please note that with Griess reagent, the lowest detectable concentration of nitrite would be 1.5 µM.

Hope that helps..

Hi Sagar,

Here is the protocol I use for BV2 or primary microglia cultures:

NO assay

Prepare Griess reagent:

2.3ml Phosphoric acid (85%)

1g Sulfanilamide

0.1g Naphtylethylenediamine

97.7ml water

Vortex. Prepare Griess reagent at least half a day before use. Protect from light.

Keep media from cell cultures on ice.

Prepare standards from sodium nitrite (NaNO2): from 0 to 1000 µM. Use the culture media for dilutions.

Pipette media and standards 50µL/well on a 96w-plate.

Add 50µL of Griess reagent on top of each sample. Shake for 10min and protect from light.

Measure absorbance at 540nm in the reader machine.

Good luck,

Sighild

Sighild, I am struggling to get reproducible results with the Griess reagent on BV2 cells. I noticed you prepare the Griess reagent several hours in advance, while I usually prepare it fresh, which might explain the inconsistency in my experiments. Have you tried preparing the Griess solution fresh without being able to measure NO after stimulation?

Could you give more details about the culture itself, please? I would most greatful.

ex.,

Inflammatory stimulus (time and concentration)

Medium (type, supplementation, etc)

Thank you!

Hi,

We used griess for a few weeks even (never fresh, but it should work fresh too). LPS 100 ng/ml or more, up to 1 ug/ml, for 24 or 48 hours work really fine. Media was DMEM F12.

Hope it helps,

S.

Thank you Sighild Lemarchant 

Hello, I have the problem that my griess reaction standard curve doesnt work because it always gets yellow at high concentrations and deep purple. Can you please help me.

Thank you!!! I'm goning to beging NO cuantification 

after preparing sodium nitrite solution, when we have to add Griess reagent and have to take absorbance?

Hi Ummay,

Add the Griess reagent (A+B) in both standards and samples subsequently. Keep it for 15-20 min at RT and take readings.

Good luck!

Hi Ummay,

I have done the same procedure but found that absorbance continuously increased. can u help me to sort out the same.

Hi everyone,

If I am preparing the stock solution of the sodium nitrite, should I use the culture media or deionized water to dissolve it? I know I should be using the culture media when making the dilutions,but how about the stock? Thank you in advance.

@Lilli Sester, did you solve this problem that you encountered and how?

Thank you in advance!

Mix the equal ratio of Griess reagent (1% SA and 0.1% NEED) and sample. Then incubate in dark for 15 min at room temperature. The absorbance of the reaction mixture is measure at 540 nm.