2.5. Nitric Oxide Synthase Activity Assay

RAW264.7 murine macrophages were cultured in VLE-DMEM medium (Biochrom) supplemented with 10% FCS at 37 °C, 5% CO₂, 95% humidity. At about 80% confluency cells were stimulated for 24 h with 20 ng/mL LPS and 20 ng/mL murine IFNγ to stimulate the expression of inducible nitric oxide synthase (NOS2, iNOS). NOS activity was assessed using a colorimetric Nitric Oxide Synthase Activity Assay Kit (ab211083, Abcam, Heidelberg, Germany) according to the manufacturer’s instructions. Briefly, stimulated cells were washed 3 times in 1× PBS, harvested in NOS assay buffer supplemented with Protease Inhibitor Cocktail (Roche, Mannheim, Germany) and centrifuged for 10 min at 4 °C at 10,000× g. The supernatant was collected, and protein concentration was determined using a BCA-based assay with a BSA standard curve. Compound 6 (3, 10, 30, 100, 300, 1000, 3000, 10,000 µM) or DMSO as vehicle was mixed to the protein extract (50 µg) and incubated for 10 min at room temperature before adding a NOS master mix (containing cofactors, substrate and 10–12 µM BH4), incubation at 37 °C for 30 min and subsequent analysis of the colorimetric product at absorbance of 562 nm.