Nitrite measurement - Help needed! - (Jun/12/2006 )
We are constantly facing problems with the measurement of nitrite in cells (Macrophage and Neuro2A) activated with LPS. The problems include erratic values, dispersion of duplicates and faible values. This is surprising as this experiment with Griess reagent and especially using macrophage cells are very commonly seen in research articles.
The experiment is
1. Seeding of cells in 96 wells at 4E4 cells/well (macrophage ) and 8E4 cells/well (N2A) in 200 µL of 10% FCS RPMI and incubated for 24 hrs at 37°C CO2.
2. Change of medium and activation with LPS at 0.5, 1, and 5 µg/mL and incubated at 37°C CO2 for another 24 hrs.
3.Supernatant taken for Griess reagent, 10 mins after plate is read at 540 nm.
The Griess reagent works as it works on standards as low as 5 µM of sodium nitrite. LPS works as we see the iNOS in RT-PCR experiments. So can not we measure the nitrite? And why erratic values? And is it a must to use a mixture of IFN and TNF to activate the cells?
Any suggestions will be appreciated. Thanks
IRD ; UMR152 -IRD-Université Paul Sabatier Toulouse III
Laboratoire de Pharmacochimie des Substances Naturelles et Pharmacophores Redox
Have a look at using DMEM instead of RPMI. The reason is that RPMI has mM amounts of Nitrate and there maybe contaminating amounts of Nitrite that is masking your Macrophage NO. We have always used DMEM for our J774 murine macrophage cell line because we have also wanted to measure NO3- in the media via Nitrate reductase assays.
Try also to increase the number of cells at seeding, you may have positive results at RT-PCR, but it maybe below the level of sensitivity of the assay. Chemiluminscence detection via a Analytix NO detector will give you level of NO2- of 100-200nM, which is far below the Griess sensitivity.
I'm agree with Rhombus. try also to perform Griess assay in cuvette to ensure that sensibility of your assay is the same of microplate.
Thank you guys for your replies . Regarding:
-changing medium from RPMI to DMEM. Well its true that we work with RPMI to culture the cells in flask and also in the 96 well plates. However, before introducing the toxin, the medium in the plates is changed from RPMI to EBSS, washing the wells once with 50 ml of PBS. The plate is further incubation with EBSS + LPS for 24 hrs. It does not help...Maybe the cells are stressed by the change to EBSS...???
-we have already worked with higher cell concentration...no change either.
-the Griess reagent we use is sensitive to 5 µM of nitrite using the standard curves.
Using the other techinque that you have mentioned Rhombus is quite expensive thats why opted for Griess reagent. However we will give the usage of DMEM with higher cell concentration a go. Thanks again.
Why are you using EBSS for your 24 hr LPS stimulation ? You should use DMEM for the 24hrs LPS stimulation, I am sure that this will cure your problem
The only other thing that could be happening is that if there is a small Endotoxin contamination from another source i.e. your FCS/media, this can sometimes cause Tolerence.... i.e. a dose of LPS
that should induce iNOS, will after a tolerating predose inhibit this process.
In reply to your question as to why I was using EBSS for LPS stimulation....I read a publication where they used replaced DMEM with Hank's Balanced Salt Solution to maintain their plated Hepa1c1c7 cells just before adding the stimulator TNF after which the HBSS was collected, and stored. The amount of nitrite was then determined by Griess reagent [J. Pharm. Pharmaceut. Sci., 6(2):302-307,2003.] .
As I know NO radical can readily react with the components of the medium, I preferred to use Earle's Balanced Salt Solution (EBSS). However, in accordance to your suggestion with the tolerence ..my data with LPS (in both EBSS and RPMI) shows that instead of production of NO, there seems to an inhibition as my OD values are near zero or even less. So there might be contamination as last RT- PCR experiments with non-treated cells show iNOS.
Anyhow, we are now changing from RPMI to DMEM...keeping fingers crossed.
Make up fresh LPS for every experiment i.e weigh out LPS and dissolve in DMEM. Is the batch of LPS new ? it might have gone off ?
There should be 30-50uM Nitrite in your DMEM after 24hr stimulation.
We have been doing this for 20 years, I used to make iNOS from J774 for Wellcome research lab for Professor Salvador Moncada and Sir John Vane , you may have heard of them, one in fact won a Nobel Prize for Physiology/Medicine.
Sorry can not think of anything else to help you, good luck
As suggested we changed the medium from RPMI to DMEM. We saw that our Neuro 2A cells do very badly in DMEM in plus we did not see any difference with Griess reagent.
Then we increase the cell number to 1.2 E6 cells/well. Here we see a very slight improvement with nitrite content calculated at 1 µM from the standard curve. We have used lab-make and a commerical kit (Roche).
I am wondering if the stimulatton of cells with a cocktail of cytokines like Interferon and TNF in addition to the toxins, is necessary to dose nitrite content as I often read in the articles.
I have not used your specific cells but in the past have looked at cells that require cytokine stimulation and well as LPS stimulation.
We have used a cocktail that includes IFN Gamma, IL1, IL6 and TNF. There are plenty of papers in the literature that will give you an idea about the concentrations required.
Have you tried a Nitrate Reductase to reduce Nitrate back to Nitrite before doing the griess assay. It maybe that your cells produce oxidising radicals which push the nitrite to nitrate ?
Sorry but I am running out of ideas to try and help.
We have the same problems in our lab. Some batchs of macrophage(we used RAW264.7) can produce detectable nitrite after LPS stimulation, but others can't. We use DMEM medium and we usually seed 200ul cell in 96 well microplate with a density of 2*10^5 cell/ml. Do you know why it happened?
I also try to stimulate nitrite from caco-2 cell using cytomix(IFN-r, TNF-a and IL-1b). A lot of papers work on it, but I still can't not get the detectable nitrite after stimulation with cytomix. I wonder if you did that before and do you have any suggestion for it?