# Constituents from _Ageratina pichinchensis_ and Their Inhibitory Effect on Nitric Oxide Production
## 2 Materials and Methods
### Anti-Inflammatory Assays
#### 2.3.2 In Vitro Anti-Inflammatory Activities  
The in vitro anti-inflammatory activity was evaluated using the in vitro model of nitric oxide inhibition of the RAW 264.7 macrophage cell line. For this purpose, the cell line was cultured in 75 cm\({}^{2}\) flasks; subsequently, the cell viability was determined by MTS, applying the treatments (pure compounds at different concentrations) to determine whether the compounds inhibit cell viability. Next, macrophages were cultured in 96-well plates, and their nitric oxide inhibition was determined using the supernatant, with which the Griess reaction was performed, measuring nitrite production as an indicator of NO production in the medium, since NO is unstable under aerobic conditions. The cytokine IL-6 was determined using the PEPROTECH\({}^{\text{TM}}\) kit, while NF-kB activation was evaluated in macrophages of the RAW-Blue\({}^{\text{TM}}\) cell line. Macrophage Culture in 75 cm\({}^{2}\) Flasks  
The ATCC\({}^{\text{\textregistered}}\) Tib-71\({}^{\text{TM}}\) macrophage cell line (Georgetown, Washington, DC, USA) was cultured in the ADVANCED DMEM/F12 medium (GIBCO) + 1% GLUTAMAX\({}^{\text{TM}}\), supplemented with 3.5% fetal bovine serum (FBS) without antibiotics, in 75 cm\({}^{2}\) cell culture flasks, then incubated in 5% CO\({}_{2}\) atmosphere at 37 \({}^{\circ}\)C until 70%-90% confluence was reached. After that, the cells were detached, and 10 mL of the culture medium was applied and centrifuged at 9500\(\times\)\(g\) for 5 min. The cell pellet was resuspended in 5 mL of culture medium, and cells were counted using a Neubauer camera for subculturing in 96-well culture plates. Cell Viability of RAW 264.7 Macrophages  
Macrophages were seeded in a 96-well plate (10,000 cells/well) with 0.1 mL of ADVANCED DMEM/F12 + 1% GLUTAMAX\({}^{\text{TM}}\) culture medium with 3.5% fetal bovine serum and incubated for 24 h at 37 \({}^{\circ}\)C in a 5% CO\({}_{2}\) atmosphere. After the incubation time, the compounds **(1-11)** were dissolved in dimethyl sulfoxide (DMSO) and filtered through 0.45 \(\upmu\)m cellulose membranes, and the compounds were added at different concentrations. The vehicle DMSO at 0.5% _v/v_ and etoposide at 40 and 20 \(\upmu\)g/mL were incubated for 20 h [32; 33]. Then, 20 \(\upmu\)L of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethyloxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reagent (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay PROMEGA) was added to each of the wells and incubated for 4 h. Finally, the optical density was measured at 490 nm in an ELISA plate reader (Epoch microplate spectrophotometer, Bio-Tek, Santa Clara, CA, USA) [49; 50]. Treatment of Macrophages with Compounds and LPS  
Macrophages (30,000 cells/well) were seeded in a 96-well cell culture plate with 0.2 mL of ADVANCED DMEM/F12+ 1% GLUTAMAXTM culture medium with 3.5 fetal bovine serum and incubated for 24 h in a 5% CO\({}_{2}\) atmosphere at 37 \({}^{\circ}\)C. Subsequently, the compounds were applied at 150, 75, 37.5, 18.8, and 9.4 \(\upmu\)M; indomethacin (positive control) at 85 \(\upmu\)M; and the vehicle DMSO at 0.5% and incubated for 2 h. Next, 2 \(\upmu\)g/mL of lipopolysaccharide-LPS was added and incubated for 24 h. Finally, the supernatant was removed by placing 50 \(\upmu\)L of each well in an ELISA plate for the Griess reaction, and 100 \(\upmu\)L of supernatant was reserved for the cytokine analysis. Determination of NO in RAW 264.7 Macrophages  
Next, 50 \(\upmu\)L of each previously obtained supernatant was used and mixed with 100 \(\upmu\)L of Griess reagent (50 \(\upmu\)L of 1% sulfanilamide and 50 \(\upmu\)L of N-(1-naphthyl) ethylenediamine dichlorohydrate 0.1% in 2.5% phosphoric acid) for 10 min. Finally, the optical density was measured at 540 nm with a plate reader, and the concentration of nitrite present in the samples was calculated by comparing it with the optical density of a NaNO\({}_{2}\) standard curve prepared in a fresh medium [33; 34; 44].