Hi Jeremy Mcguire

To estimate the amount of NO produced by cells. You can use Griess reagent, which can be prepared by yourself in your lab. Griess reagent consist of 0.2% naphthylethylenediamine dihydrochloride, and 2% sulphanilamide in 5% phosphoric acid. Prepare both the compounds separately in an amber colored bottle label it and store it in 4 C. Mix equal volume of both the solutions before estimation.

To estimate the NO from cell culture supernatant, add equal volume of supernatant and Griess reagent and read the pink color developed at 540 nm using a multiplate reader immediately (stability of this reaction is very less hence read the plate with in 10 min).

Note:

The stability of Griess reagent itself in very less hence prepare the reagent freshly every time however it has self life for nearly a week when stored at 4o C

Do not mix both the reagent together because the induce auto oxidation hence cannot be store for long time also once mixed it has to be used within half an hour.

Best use of the reagent is always preparing them in your lab instead of buying the readymade kit.

All the best

Hi Jeremy

No, you cannot use colored media as color developed after the addition of Gries's reagent is same as the color of the phenol red containing media. I would suggest you use colorless media. If you are new to NO estimation, I would suggest you to buy the kit. Hope this will help

You can use DAF-2 DA fluorescence assay

You will be able to do both microscopy and flow cytometry with this assay

Some useful links:

http://www.sigmaaldrich.com/catalog/search?interface=All&term=daf-2+da&lang=en®ion=IN&focus=product&N=0+220003048+219853147+219853286&mode=match%20partialmax

http://jxb.oxfordjournals.org/content/57/12/3043.full

http://www.ncbi.nlm.nih.gov/pubmed/15380680

I agreed with Aslam Khan. If you decide to use Gries method to measure NO, the media must be phenol-red free. We used the kit from Cayman, it worked well.

If you have a license to use radiolabeled arginine, the enzymatic conversion assay is very reliable, and not prone to the issues related to spectrophotometric assays. The Dowex 50 resin is good at binding most of the excess arginine, but you still need to control for non-specific conversion to other non-binding metabolites (i.e., ornithine from the arginase pathway)

The Griess reaction measures nitrite, not authentic NO. There are a lot of papers that do not appreciate that nitrite reflects NO production rate and not NO concentration. sGC is activated based on NO concentration (few nM/L) not based on nitrite concentrations (few micromolar).

If you use DAF you must take into account that this may react unspecifically with N compounds others than NO (http://jxb.oxfordjournals.org/content/57/12/3043.full)

But as an estimation of NO contents it may be a good approach. The Griess assay explained by Sundarajan Kannan is quite nice and easy to develop as well.

Good luck!

You can use DAF with the appropriarte controls such as using cPTIO as a scavenger of NO to ensure that the signal observed is due to NO.

Good luck!

Hi

You can use Griess reagent from sigma to determine NO production. And I agree with Aslam Khan too. The Griess method is very easy and fast. After incubation of your cell line with your treatment, take out the media to 1.5 ml test tube, centrifuge 300 g for 5 min. take 100 µl of the supernatant to 96 well-plate, add 100 µl of Griess reagent, incubate at RT for 10 min and obsorbance at 540 nm. Before that, make the standard equation with various concentration of standard Sodium Nitrite by using the same manner. Apply the OD540 value of test sample to the standard equation, you can get the answer.

Good luck!

Hi, Jeremy, you can use Hemoglobin assay for NO quantification. But you have to use medium without phenol red. DAF2-DA you can use only for localization of the sites of NO-accumulations, but not for the measure of NO-production. It is difficult to made any conclusion form DAF2-DA because this is not ratio-metric dye.. Good luck!

I agree with David, who importantly pointed out that the Griess reagent, although commonly used and relatively simple, measures nitrite (a decomposition product of ˙NO). Nitrite can be further transformed to nitrate, so Griess assay may underestimate the true levels, unless you add nitrate reductase to your samples and standards, prior (1hr) to the Griess reagent. The most direct way of measuring ˙NO would be to use an ˙NO-specific electrode.

If you need to "quantify" NO produced in your cells, DAF-2-FM is not what you need. You can detect changes in the amount of NO synthesized, but you can't quantify it. The colorimetric assay is a better approach, but if you have access to them, I would recommend the use of the NO electrode or the NOS activity assay (this last one uses radioactive L-arginine). Be aware that the half life of NO is only 4 seconds, and its detection may be difficult. I suggest in case of the NO electrode that you serum starve your cells (I go down to 0.5% FBS with ECs) and that while you perform your analysis you use PBS with very low amount of FBS for better readings. Hope this can help. Good luck!

I agree perfectly with Vinod Damodaran that the most reliable and specific method to quantify nitric oxide (NO) available to date is the ozone based chemiluminescence method. David, Griess’ assay is an indirect method which measures nitrite only. The reproducibility of the Griess assay is not so wonderful, so if you are able to have access to the ozone based chemiluminescence method please go ahead to use it. Follow the links provided by VINOD.

Yes you can use DAF, DAR, Griess assay and even by electrode

Hi Jeremy, we also use the Griess reaction to measure NO production in endothelial cells. It works well in most cases, but in some experiments the assay is not sensitive enough. We then shift to the measureIT Nitrate kit from Invitrogen. This assay is based on the same Griess Reaction, but is a fluorescent assay that has greater sensitivity compared to the regular griess protocols. cPTIC works well as an scavenger of NO in these assays and should be used as control in my opinion.

Hi Jeremy!

I want to add to some of the above. First, we do not recommend use of the NO electrode. This requires a long lasting expertise. Also please refer to the papers of Miriam Cortese-Krott, who is in my friends list for recent advances in the DAF method. THis has of course certain limitations but will indeed give you a signal under the microscop and in FACS.

The Cayman kit with Griess reagent, i cannot recommend. It naturally measures nitrite and nitriate must first be reduced to nitrite in order to be measured. Nitrate is produced when NO reacts with heme globins. The reaction from nitrite to nitrate is actually very slow and does hardly occur in vivo.

To quantify NO , I actually would recommend EPR spectroscopy or chemiluminescence, this is at least what we use right now.

There is no specific advantage of any of the methods; using an NO scavenging method has the disadvantage that you look at the little left-overs of NO wherever you take you sample (e.g. outside a cell) and then try to extrapolate how much it was at the biologically relevant site (i.e. within a cell). This can be orders of magnitude off and is highly influence by the medium or other artefacts. Using a method which assays break-down products of NO has the advantage to be able to fully capture NO synthesis but you cannot make a statement whether at you biologically relevant site its was actually NO. For example under oxidative stress, total NO (nitrite + nitrate) will by and large not change because NO synthesis did not change. If you measure NO, you would get reduced NO signals but then cannot differentiate whether it was reduced NO synthesis or increased breakdown. So there are horses for causes. You have to define your scientific question well and may even need to use different methods to measure NO, nitrite and nitrate. A little technical issue when measuring nitrite/nitrate, make sure you use ultra-pure water that has very low basal levels of nitrate/nitrate. Milipore water often has high (!) levels!

Nitric oxide is generated by NOS and then undergoes reactions with oxidents, mostly oxygen and superoxide; thus you build up nitrite and nitrate in you culture media.

Firstly the two most common assays measure only nitrite, so one must convert the nitrate to nitrite. Typically people use the enzymatic method, but I love the direct chemical approach. Buy some Cadmium powder, −100 mesh, from Sigma. Cut the bottom 5 mm off an eppendorff tube and stick a needle through the side and you have a little scoop. You place about 5% w/v of the fine, toxic, powder into you tube.

The powder has a huge surface area and converst the nitrate to nitrite. You can spin after an hour and all the Cd goes to the bottom and you can remove the supernatent.

To make sure that you media isn't affecting the reduction I recomend that you spike control media with nitrate and make sure that it is all convered to nitrite in about 1 hour. The V(III) method in 'A rapid, simpl spectrophotometric method for simultaneous detection of nitrate and nitrite by Miranda et al., 2001 isn't bad at all.

Finally, use the 2,3-diaminonaphthalene (DAN) method instead OF Griess if you have low levels. The paper by Misco

http://www.sciencedirect.com/science/article/pii/S0003269783714491

covers everything, but I prefer to strip out the protrin with a Microcon microconcentrators, 30 000 MW cut-off in a microcentrifuge

I exclusively use a Sievers NO analyzer for ozone based chemiluminescence. I have tried Griess and found it wasn't sensitive enough for my needs. With the Sievers I can get measurements at picomole levels. Depending on how you set up the reducing agent for the assay you can measure nitrate, nitrate/nitrite, and even capture data on SNOs. DAF I don't like as much because of the need for multiple controls to understand what exactly you are measuring.

Some tips no matter which assay you choose:

USE dH2O DIRECTLY FROM SOURCE. Do not use dH2O that has been sitting around or use any buffer that has been opened previously. Aqueous solutions will absorb atmospheric nitrogen compounds that will give you high background in many of these assays resulting in significant sources of contamination affecting reproducibility. Prepare fresh standards from highly concentrated stocks of nitrite/nitrate as well, for the same reason. Do not reuse standards day to day. If using test tubes at any point that will contact supernatants or standards - rinse them extensively in dH2O right out of the distilled tap before hand. Even sterile unopened tubes will contain significant amounts of contaminating nitrates that may be detectable. I even go so far as to rinse my pipette tips several times in fresh dH2O prior to allowing them contact with my samples. Depending on the range you are measuring this may be overkill, but if you want to really make results reproducible these steps will help. Definitely necessary if you are after uM or nM measurements.

Be aware that in 96 well plates that the outer wells will absorb significantly more atmospheric nitrogen compounds than the inner wells. To prevent this in 96 well plates I only use the inner wells and I place replicates in such a way so that they have the same orientation from group to group with respect to distance from the center and edges so that these effects will average out. You can fill the remaining wells with media as a sort of barrier to gas exchange (don't know if it works, but my results are good so I keep doing it!) Another option is a gas permeable stick-on lid. This won't prevent absorption of contaminants, but at least background levels will be uniform across the plate. Depending on whether or not you are in a city, and whether you are after mM, uM, or nM levels, the atmospheric levels of contaminants may or may not be a big problem but all of these steps can help. Good luck!

Dear,

I have worked with NO measuring methods at the last years and I strongly recommend that if you will choose a coloremtric-based assay you should use the nitrate reductase assay or some that convert the nitrate in nitrite followed by Griess reaction. As example you can see: Trypanosoma cruzi: effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on levels of cytokines, nitric oxide and iNOS expression in cardiac tissue in the acute phase of infection in mice from Panis C, Mazzuco TL, Costa CZ, Victorino VJ, Tatakihara VL, Yamauchi LM, Yamada-Ogatta SF, Cecchini R, Rizzo LV, Pinge-Filho P.

Exp Parasitol. 2011 Jan;127(1):58-65. doi: 10.1016/j.exppara.2010.06.030.

I am available if you have any further question.

Good luck!

Carolina Panis

You may use FL2E which is an NO specific fluorescent dye from Strem Chemicals. (link: http://www.strem.com/catalog/v/96-0396/33/nitric_oxide_sensor_intracellular_kit_no-on_fl2e). Does not have the same issue as DAF. It might pick up nitrosothiols, so you would have to figure out a way of blocking thiols at the same time as you loading the dye.

Can anyone plz recommend a commercial laboratory that analyzes blood samples for NO, prteferably in the West or Midwest? Thank you

Hi Jeremy,

You have a lot of competent advices, I am agree with comments of Matthias Totzeck and Harald Schmidt.

The methods that you are going to use always will give you information that you have to interpret in appropriate way. When scientists use Griess reaction for measurements and quatitation of NO production, they normally use quite long-time cell preincubation (with ionophore for example) due to limitation of sensitivity, what restricts a physiological significance of study.

But the most important, that difference in nitrite/nitrate levels between conditions will not reflect NO bioavailability, as often indicated. It could give you difference in potential NOS activity, but oxidative products of NO (peroxynitrite, for example) also are converted to nitrite/nitrate depending on pH and its multiple targets in cells, and will not reflect the functional NO availability.

If you have access to the EPR spectroscopy (NO spin-trapping) or chemiluminescence, it could give you reliable instruments that you can use to study quantitative response from the conditions.

Good luck!

You can use Griess reagent assay which is based on colorimetric approach. To do that, you even don't need a kit and could prepare all the needed reagents in your lab. What you basically need is - 0.2% naphthylethylenediamine dihydrochloride, and 2% sulphanilamide in 5% phosphoric acid. And yes, you could use the colored media, like DMEM.

Hi geremy

you can try Griess assay for NO estimation. I did this assay for my experiments.I got good results.

Hi Rony, Can you write the detailed protocol with reference for Griess assay. I have to do it for NO. Thanks.

You should first prepare the two solutions and keep them separated, while NED should be kept in dark. Just before making the measurement, mix the solutions by adding part of NED to part of Sulphanilamide. Add 100uL of the mix to 50uL of the medium of the interest and mix well. Let it stand in dark for 5-10 min before reading. Of course you need to set up a calibration curve using 1mM sodium nitrite - make serial dilutions starting from 500uM to 0 while every next dilution will be a half as a previous one (i.e. 500, 250, 125 ect.) Establish all the needed controls as well. I was able to measure as less as 5uM of sodium nitrite in medium of microglia primary cells activated with LPS for 24h. I never used nitrate reductase for my purposes.

Hope this is helpful!

I recommend that if you will choose a spectrophotometric assay, you can use Durak I, et al. method (Acta Anaesthesiol Scand. 2001 Jan;45(1):119-22.)

Hi Jeremy,

If you know the proportion of nitrate:nitrite and that it remains constant between experimental conditions (probably unlikely) - you may back calculate using griess only - but the correct and most precise appoach is to to reduce nitrate. I do not have much experiences measuring NO using kits as I used NO sensor and other biochemical means to reduce nitrate then measure nitrite using Griess reaction. attached is a quick, cheap and very robust protocol that I would highly recommend. Regards,

Colin

Article Pathophysiological basis of acute inflammatory hyperaemia in...

Griess reagent - the 2 solutions should be kept in the dark in a fridge separately - probaly best to cover the bottles with tinfoil and label them A and B etc. Prepare solution weekly.

Thanks Rony

product of NO, was measured in the culture media by using the spectrophotometric Greiss reactio. I thinks it's better for your experiment.

You can use Griess reagent assay which is based on colorimetric approach.

Hi Mirza

would you please write whole protocol with its reference. I want to measure NO in root hair of plant. Can i use this method to do it?

Thank you sir Prof Mirza, please can I get reference for the above protocol? Thank you sir.

We can use Griess assay  to quantify the amount of Nitric Oxide in Extra and Intra cellular level. But think about NO, it's highly reactive gaseous species it will escape easily (Headspace NO) so Griess assay is not accurate assay. we can use Chemiluminescence based NO analyzer, use air tight HPLC vial and inject both Headspace and solution space and quantify the amount of NO.

DAF always create lot of problem, for this we need proper sample preparation and it's not specific for NO.

Hi Jaya

Would you please be more specific?  As you Nitric Oxide  pathway is dependent on arginine and citruline pathway. If your compounds are iNOS2 substrates then you need use rINOS2 enzyme to test effect on your compounds.

hi, Rony I need to know hoe we can calculate hoe much induction of inos is there in treated compared to control with absorbance value. Hoe to use standard value in these cases??

I did this experiment using Griess Reagent but the absorbance at 540nm was very high when RPMI-1640 medium was present, and I don't know how to solve this problem. Do I have to change the  to uncolored medium?

hi LIang, what do you mean by high absorbance, is this present in treated samples or only rpmi media shows high absorbance, pl we specific.

We can quantify NO in cells by using Griess reagent indirect measurement of NO (colorimetric based assay). If the assay is sorter incubation time we can use PBS.

Yes, Liang you have to use colorless medium with Griess's reagent. The reason for this is 540 wavelength is for red color, if you use rpmi 1640 with phenol red then you will see the high absorbance as you are picking up phenol red as well. Hope that will help.

Whatever solution (plus additives) You use to measure the concentration of NO2 in cell culture supernatants by colorimetric means (such as Griess) it is highly recomended to run a wavelenthscan prior to set up Your measurement parameters. Phenolred (PR) is anyway problematic, as is shows an absorption at 540 nm, the ODmax of the NO2-Azodye. However, it is further complicated by the fact, that each medium contains different amounts of PR, and that OD at 540 from PR depends on the pH in the well - which may be affected by copmounds You add in order to modify the NOS-activity. Thus a simple blanc substraction might not help You out. In this case it is necessary to measure at several wavelengthes. I have added a grafic that illustrates this point.

World Precision Instruments (WPI) have a range of nitric oxide specific sensors (electrodes) that can be used both in vitro and in cell cultures. They require calibration and come as a package (a Free Radical Analyser system). They are more expansive than a kit, but they work really well. 

https://www.wpiinc.com/products/physiology/iso-nop/

Hi, Can any one provide me NO production (Griess reagent), COX-1 COX-2 Procedures for anti inflammatory activity

I did experiment using griess reagent on Raw 264.7 cell line induced with LPS, samples . then incubated in 24h. but the experiment was fail. Does anyone  have any experience?

Phuong, I do this assay frequently on RAW cells. Be sure there is some serum in the media, as LPS needs sCD14 to bind to cells. You can also try removing serum-containing media and simply replacing with starve media for only a few hours (4-5) prior to LPS stimulation. If that doesn't work, try a different LPS. I've had good luck with E Coli 0111:B4.

Avoid cell culture medium that contains NO3- as a component (such as RPMI-1640) is not a suitable medium to detect [NO3- + NO2-].

I have a RAW 246.7 cell line of passage num 35. Is it suitable for LPS stimulation studies?

I would like to know if the Griess reagent assay be done with 96 well plate

You can see the following paper for Griess method of NOx (nitrate+nitrite) assay.