A couple of points:-

Are you using Human or animal cells- this is important because the ratio of Nitrite/Nitrate is completely different. Animal cells have a ratio of approximately 90% NO2-/10% NO3-. However in human cells this ratio is completely reversed. If you are using the Griess assay, which measures NO2-, then using animal cells and measuring Nitric Oxide release is easy.

What cell culture media are you using- this is very important if you are using Human cells and you have to use a nitrate reductase assay in order to measure Nitric oxide release. RPMI has mM amounts of NO3- and will swamp any levels of Nitric oxide release from cells. DMEM has undetectable levels of nitrate. You will need to dialyse your FCS/FBS to get rid of NO2- and NO3-

The levels of NO release are completely different between human and animal cells- animal cells in general release far greater amounts of NO. Therefore it is a lot easier to measure NO2- with the Griess reaction.

Purchase your own chemicals and make your own solutions. The chemicals will last for decades and therefore you can make fresh solutions and not worry about use by dates

A) Griess Reagent containing: 1) 0.05% (0.5mg/mL) N-(1-naphthyl)ethylenediamine dihydrochloride; 2) 0.5% (5mg/mL) sulfanilic acid; and 3) 2.5% phosphoric acid

I hope this is useful

Kindest regards

Neale

Hi Elisa Avokio

Please find this link which is similar to your question and many international experts given may different approachs for estimating NO. I hope this may be helpful, if your substance is interfere with the reaction perform a blank run containing all the substance except griess reagent/ only protein and griess reagent/ all other substance except protein etc and try to corrolate your results

https://www.researchgate.net/post/Nitric_Oxide_Assay2?ev=hdr_person_add_comment_question_target

All the best

https://www.researchgate.net/post/Nitric_Oxide_Assay2?ev=hdr_person_add_comment_question_target

Thanks a lot for your help Sundarajan!

Best,

Elisa

A simple Saville assay should be rapid enough to detect nitroso products. The Promega Greiss kit should be ok in my opinion. Just avoid the destabilizing components in your buffers/media. The Promega techbook has all details http://www.promega.com/~/media/Files/Resources/Protocols/Technical%20Bulletins/0/Griess%20Reagent%20System%20Protocol.pdf

I always used the promega kit.

it is really great, easy and fast!

If you have any question about the kit or anything else just let me know.

best,

Sebastian

A couple of points:-

Are you using Human or animal cells- this is important because the ratio of Nitrite/Nitrate is completely different. Animal cells have a ratio of approximately 90% NO2-/10% NO3-. However in human cells this ratio is completely reversed. If you are using the Griess assay, which measures NO2-, then using animal cells and measuring Nitric Oxide release is easy.

What cell culture media are you using- this is very important if you are using Human cells and you have to use a nitrate reductase assay in order to measure Nitric oxide release. RPMI has mM amounts of NO3- and will swamp any levels of Nitric oxide release from cells. DMEM has undetectable levels of nitrate. You will need to dialyse your FCS/FBS to get rid of NO2- and NO3-

The levels of NO release are completely different between human and animal cells- animal cells in general release far greater amounts of NO. Therefore it is a lot easier to measure NO2- with the Griess reaction.

Purchase your own chemicals and make your own solutions. The chemicals will last for decades and therefore you can make fresh solutions and not worry about use by dates

A) Griess Reagent containing: 1) 0.05% (0.5mg/mL) N-(1-naphthyl)ethylenediamine dihydrochloride; 2) 0.5% (5mg/mL) sulfanilic acid; and 3) 2.5% phosphoric acid

I hope this is useful

Kindest regards

Neale

Hi!

I had performed the NO assay without using ELISA kit. What I did was, I challenged the cells with a stimulus, which is LPS for 24 hours and measure the production of NO in the supernatant. There are a few factors you need to take into consideration:

1) Number of cells - maybe the number of cells you are using is not enough, hence, NO production is too little for detection.

2) Type of media - a non-phenol red media is use instead of phenol red, because if NO is detected in supernatant, the supernatant will turn into pink after Griess reagent is added.

3) Incubation hour - Depends on the type of cells you are using. I recommend you to do a time-course to determine the optimum hour for NO production for your type of cells.

Hope this helps. :)

Good luck and all the best!

Thanks a lot to all of you for your answers!

I'm using human cells and I have different media: M199 and DMEM+DMCB201; I'm filtering the supernatants with 10K cut-off filters, to exclude serum proteins.

My samples have been frozen to -80°C immediately after collection, I hope that this passage has not damaged them.

I will take into account your suggestions and I will repeat the measurements.

Thanks a lot!

Best,

Elisa

Hi Elisa,

I have measured NO in a range of biological fluids (serum, saliva, urine) but not in cell supernatants. However, the protocol I have used should work well from what you have described. I have used a conversion step (nitrate to nitrite) for 30 mins followed by Griess reaction - the reductiion stem is chemical and not with reductase. Advantage of my method is low cost (reductases can be expensive), quick and reliable with high conversion rates (between 98-100% conversion of nitrate in 30 mins). best of luck!

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Obviously if you have pink medium you would have problems with spectrophotometer in measuring Griess. However, if you produce enough nitrate/nitrite, youl will be able to dilute your supernataant enough so as not to interfere with spec reading

Hello Neale, 

can you please explain the discrepancy in the difference in nitrite and nitrate ratio between human and animal cells..?