Griess reagent

RAW 264.7 cells were seeded in a 96-well microplate at 3 × 10⁵ cells/well with complete media. The cells were allowed grow and adhere for 24 h. The cells were treated with NQC (50, 25, 12.5, 6.25 and 3.125 µM) and negative control (0.1% v/v DMSO in water) for 4 h prior to LPSEc (1 µg/mL) and allowed to incubate for 20 h at 37 °C, 5% CO₂ incubator. Nitrite exist as a stable metabolite of nitric oxide, thus nitrite present in the supernatant was quantitatively measured as a chemical marker of nitric oxide (NO) production using Griess reagent (Promega, USA; 0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 2.5% phosphoric acid). This assay was carried out following the manufacturer’s protocol where 100 µL of cell supernatant sample was incubated with 100 µL of Griess reagent at room temperature for 10 min and the absorbance at 540 nm was measured using Molecular Devices Spectramax M3 Multi-Mode microplate reader. A standard curve of sodium nitrite was plotted and used as a reference for extrapolation to quantify the amount of nitrite present.

RAW 264.7 murine macrophages were seeded at a density of 2 × 10⁶ cells/well in 6 well cell culture plates (2 mL/well). After 24 h, the medium was removed, and cells were washed with Dulbecco’s phosphate-buffered saline (DPBS; Welgene). Macrophages were then treated with different concentrations of HyA (10 and 100 µg/mL) for 1 h, after which they were exposed to 100 ng/mL LPS (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Supernatant media were then collected, and nitrite was measured using the Griess reagent (Promega) according to the manufacturer’s instructions. Briefly, culture supernatants (50 µL) were mixed with 50 µL of the Griess reagent and, after incubating at room temperature for 7 min, their optical density at 540 nm was measured in a microplate reader (Molecular Devices). NO levels were determined directly from standard curves, prepared using serial dilutions of a 100 µM nitrite (50 µL) of the Griess reagent.