Nitric Oxide (NO) Determination

This assay determines NO production based on NCL method ITA-7 (2015).⁴⁴ Briefly, murine Raw 264.7 cells were plated at a confluence of 1 x 10⁵ cells/well in 10% FBS RPMI medium (phenol red free) and incubated for 24 h at 37°C. Then, medium was replaced with fresh medium containing samples and incubated for 48 h at 37°C. After the exposure period, supernatants were collected and centrifuged at 12,000 RPM for 10 min. Nitrite (NO₂⁻) quantitative determination was carried out with the Greiss reagent as indicated in the protocol. The absorbance was measured at 562 nm and a standard curve (0.12 μM to 250 μM) prepared with sodium nitrite in complete medium was used for calculations. LPS (100 ng/mL) and DPBS were used as the positive and negative controls, respectively. A cell-free interference control was prepared and taken through all the experimental steps.