Do you get a positive reaction with the kit controls (standard curve)? If the kit is functioning properly it might be due to your LPS.  LPS from some species do not activate macrophages effectively. 

What species are you using? There are huge differences regarding NO production between species, esp mouse (lots) and human (depending on individual, might be nothing)

Thank you so much for all your answers! We are using C57Bl/6 mouse, for macrophages activation - LPS from E.coli Serotype 0111:B4 (Sigma). iNOS activity was very low as compared with control (without cells). NADPH or 1mM arginine did not change activity. We tried peritoneal macrophages in different concentrations - 200000, 500000 and 1 000 000 per well. 

Maybe worth checking the supernatant using Griess reagent? Very cheap and easy assay, and if you dont see anything there, you have a problem with the cells.

Yes we did, after 20 h incubation nothing in supernatant. What can be wrong with fresh isolated peritoneal macrophages? 

Depending on what you what to validate and establish:

For NOS expression: qPCR, Western blot

For NOS activation: phospho-NOS WB, NO measurement (DAF-FM)

For activity measurement: NO (DAF-FM), nitrite (Griess assay)

For some reasons, DAF-FM is not recommended for imaging, because it takes a wash step and about extra 15-20 min for the fluorescent product to be oxidized into fluorescent form (it is not exactly real-time). 

If you really want to image NO, write to the chemists who synthesize such probes:

http://www.nature.com/nchembio/journal/v2/n7/abs/nchembio794.html

The probe is actually purchasable, though expensive:

http://www.strem.com/catalog/v/96-0396/33/nitric_oxide_sensor_intracellular_kit_no-on_fl2e_cell-trappable_no_fluorescent_probe

The drawback of this probe is that you need to charge it with copper (II) before use. Introduction of metals in the assay system potentially can have unintended drug effects. 

Thank you. We ate looking for NOS activity and NO level measurement and using DAF-FM.  LPS from E.coli did not activate macrophages effectively but now it works better with interferon activation. 

Yes, the conventional (and effective) treatment to activate macrophages into NO producing machines is LPS (200-500 ng/mL) plus interferon-gamma (IFNg, 50-100ng/mL) in cotreatment. It takes 4 h normally for iNOS protein to first appear and peak around 8-16 h. It is known in immunology that if you use LPS or IFNg alone, the effects are much weaker. For E.coli., MOI 5-10 would be effective enough. cheers 

See some related works by our group and collaborators on NO:

Article Molecular Imaging of Peroxynitrite with HKGreen-4 in Live Ce...

Article Nitric oxide as an antimicrobial molecule against Vibrio har...

>Yes we did, after 20 h incubation nothing in supernatant. What can be wrong with fresh isolated peritoneal macrophages? 

I am not familiar with in vivo or ex vivo work. Could it be a case of  (LPS) tolerance, since the animals are constantly exposed to microbes in the gut?

Another thing is make sure your treatment system has enough L-arginine in supplement. Physiological L-Arg level is 0.6 mM. (You can prepare a stock of exactly 0.6 M L-Arg in MilliQ or PBS, with some mild warming). 

***

Previously we have tried primary BMDMs as a macrophage model for inducing NO. The primary macrophages (basically immature when first isolated) takes one week's differentiation with M-CSF into mature macrophages, before challenge with LPS plus IFNg. The results were nice. You can check this out in the supporting info of our paper for peroxynitrite imaging:  

Article Molecular Imaging of Peroxynitrite with HKGreen-4 in Live Ce...

Thanks a lot Dr. Wong. We got  7-fold increase of NO level after 

LPS (50 ng/mL) plus interferon-gamma (IFNg, 20 ng/mL) treatment.