4.10. Griess Assay—Measurement of Nitric Oxide Concentration

The Griess colorimetric method was used to measure the concentration of nitric oxide (NO) secreted by RAW 264.7 cells treated with crude C. majus latex and the tested fractions. The Griess reagent (Sigma-Aldrich) is a solution of sulfanilic acid and α-naphthylamine in acetic acid, which changes the color of the solution to pink or red when there is a reaction with nitrites (NO²⁻).

To test the concentration of NO secreted by RAW 264.7 cells treated with crude C. majus latex (S1) and the tested fractions (S2, S3), cells were seeded in a 96-well plate at 5000 cells per well. Appropriately seeded cells were incubated overnight at 37 °C and 5% CO₂ concentration. The medium was then removed and the crude latex diluted in DMEM and the test fractions (100 μL/well) were administered. Wells with cells in which only the DMEM was replaced with fresh medium were used as a negative control. The positive control consisted of wells with cells treated with lipopolysaccharide (LPS) at a concentration of 100 ng/μL, which induces an inflammatory response and stimulates NO secretion by macrophages. Cells were incubated with various concentrations of the tested samples (S1, S2, S3) for 48 h at 37 °C and 5% CO₂ concentration. Then, 50 μL of medium from all test wells was transferred to a new 96-well plate. The “blank” samples were wells containing medium transferred from wells containing no cells. The empty wells were filled with NaNO₂ standard in volumes of 10, 20, and 40 µL in duplicates, which were then filled up to 50 µL with DMEM. Subsequently, the Griess reagent was administered in the ratio of 1:1 (v/v) to all wells with the tested samples and to the “blank” samples. The prepared plate was protected from light and incubated for 15 min at RT. After this time, the absorbance was measured at 540 nm using a Synergy™ H1 reader (BioTek). The NO concentration in the trials was calculated based on the NaNO₂ standard curve. The measurement of NO concentration using the Griess method was performed simultaneously with cytotoxicity analysis of crude latex (S1) and its fractions (S2, S3) against RAW 264.7 cells.