Please suggest to me an appropriate protocol for performing NO assay using Greiss reagent. I do not find difference in absorbance reading of control samples and test samples. What can be the possible reason?
- 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. I like to prepare the standard curve in the same media I used for cell culture.
But maybe the problem is not the Griess assay.
1. Which macrophages are you using? My protein never induced NO production in thioglycollate elicited macrophages only in BMDM.
2. What is your positive control? The best one is LPS+IFN
I am using Raw macrophages and LPS as stimulant, which should induce more NO production with increasing time (starting from 1 h upto 12 hrs). I am using standard NaNO2 curve (100uM onwards with serial dilution upto 1.25uM). I observe that control cells (without stimulation) show same absorbance as that of treated.
I further perform bradford assay for total protein estimation and then normalize nitrite from each sample with total protein. Is this the correct way to proceed?
I've never checked total protein in the medium because of FBS. Are you using serum free media? If your medium has FBS all you'll be quantifying if the serum. I don't know for RAW cells but I believe you should extend your time periods. NO is not pre stocked inside the cells, it needs to be produced and until iNOS is expressed it takes at least 4 to 6 hours. My NO quantifications were mada always after 24 and 48 hours. Another thing, are you freezing your supernatant? Some people say it's ok but I always prefered to assay NO right away before freezing.
Hi Mansi,
A few pointers that may help you:
1. I agree with Andre that nitrite estimation post LPS stimulation is usually between 24 and 48 hours. I myself did it at earlier time points but could not detect much nitrite.
2. The problem with this time point, however, is RAW cells divide every 12 hours. So, theoretically, at 24 hours you will have half the cells at 24 hour stimulation and the other half at 12 hour stimulation. So, you will have a sort of an average readout.
3. Try using a modified version of Griess assay. Right before the actual Griess assay, consider converting nitrate into nitrite by the enzyme nitrate reductase. Sigma has a wonderful kit that works like a charm.
4. If you have access to a flow cytometer, then instead of Griess assay, you could stain for intracellular NO by using DAF-DA. That may enable you to pick up NO at earlier time points.
Good luck.
Sir I am not quantifying total protein in media. I am isolating protein after lysing the cells from each sample and then estimating total protein, to normalize nitrite/mg of protein in each.
I agree that iNOS expression may take time to express (upto12-24 hrs) but we may see nNOS before that time point..Is that true?
Dear Mansi
Take a look at the article I'm attaching. They use RAW cells and quantificate NO after 8, 16 and 24h after LPS exposure. Apparently they could only detect NO after 16h but 24h was better. Also, iNOS expression (western blot) was detected after 6h and 8h of stimulus.
http://ajpcell.physiology.org/content/296/5/C1133
Adjust cell concentration to 1x105 cells/mL using complete RPMI medium.
2. Plate 1000 µL of cell suspension per well in a 24 well plate. Prepare triplicate wells for
each sample and duplicate wells for each control. Always leave 1 cell-free well per
nanoparticle concentration per plate. These wells will be used to assay potential
nanoparticle interference with the assay. This is the “Culture Plate”.
3. Incubate the “Culture Plate” 24 hr in a humidified 37°C, 5% CO2 incubator.
4. Remove culture medium and add 500 µL of study samples, controls, or medium blank to
appropriate wells. Position samples on the plate such that study samples are bracketed by
controls and blank medium.
5. Incubate the “Culture Plate” 48 ± 1 hr in a humidified 37°C, 5% CO2 incubator.
6. To a fresh 96 well plate, add 50 µL per well of reagent blank (culture medium used to
prepare calibration standards and quality controls), calibration standards, quality controls
and medium from each well of the “Culture Plate”. Load duplicate wells for each sample
and control. This is the “NO- Test Plate”.
7. In a separate tube combine equal volumes of reagent A and reagent B; this is the Greiss
reagent.
8. Add 100 μL of the Greiss reagent to each well of the “NO- Test Plate”.
9. Place the plate on a shaker for 2-3 minutes, allowing all ingredients to mix.
10. Measure absorbance at 550 nm.
This is what I use. It works well. Let me know if you have any further questions.
Is there another compound I could use as a positive control? I use LPS at 5ug / mL for 24h, but the nitrite stimulation is low.
I have optimized a protocol for the NO assay. In my case I was using 1000ng/mL of LPS and got a really good signal when I increased the time after platting before the LPS incubation and the number of cells on Raw 264.7 cells.
Hello Mansi, did you find a solution to your problem? As I am facing the exact same problem with mouse macrophages. I am stimulating with 1ug/ml LPS(E.coli 0111) but no difference between cell alone and LPS treated. I am using invitrogen Griess assay.
- Badges
- Science topic