2.2. Griess Assay to Measure Nitric Oxide Production by HD11 Cells

Nitric oxide production by HD11 cells was measured by the Griess assay [38] 48 h after stimulation. First, 50 μL of supernatants were harvested from triplicate wells and transferred to a 96-well flat-bottom plate (Corning B.V. Life Sciences, Amsterdam, The Netherlands) to measure the nitrite concentration. A 3.13–200 μM NaNO₂ nitrite standard dilution series (Sigma-Aldrich, Merck, St. Louis, MO, USA) was included to generate a standard curve. Griess assay reagents were made by dissolving N-(1-naphtyl)ethylenediamine at 3 g/L and sulfanilamide at 10 g/L (both from Sigma-Aldrich, Merck, St. Louis, MO, USA) in 2.5% phosphoric acid (Supelco, Merck, St. Louis, MO, USA). The Griess reagents were mixed 1:1 and 50 μL was added to the wells with cell culture supernatants and standards. The Griess reagents mixture turned purple upon reaction with nitrite ions in the cell culture supernatant. The optical density (OD) at 540 nm of each well was measured using a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany) to determine the nitrite concentration of each sample according to the nitrite standard curve.