The media were then discarded, the cells were washed twice with PBS and 200 μL of FBS-containing medium containing 10 μM of DAF-FM was added to each well, followed by one hour of incubation at 37 o C in 5% CO2. The probe solution was then discarded, the cells were washed three times with PBS and then fresh phenol red-free, FBS-free RPMI medium containing 300 μM DETA-NO with/without different concentrations of each tested compound was added to each well. The cells were directly imaged every one hour using the incucyte system for a period of 24 hours, with the random choice of four positions per well for imaging. The assessment of fluorescence corresponding to intracellular • NO was performed automatically by IncuCyte ZOOM via measurement of the green object count for all cells stained green with DAF-FM and their intensity with normalizing the results to the total object count per image. The same procedures were repeated in the case of iNOS-transfected HCC1806 cells, but without the use of DETA-NO.