Total nitric oxide assay kit

The NO level was quantified by using Total Nitric Oxide Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s protocol. 10 × 10³ cells were seeded in a 96-well plate for 24 h followed by pretreatment with ESEAF (0.13–1.0 μg/mL) for 2 h prior to LPS exposure (1.0 μg/mL) for 4 h. Upon the completion of the incubation period, 50 μL of culture supernatant was mixed with 25 μL NADH and 25 μL of nitrate reductase and incubated for 30 min at 37°C. This was further followed by the addition of 50 μL of Griess reagents 1 and 2 for 10 min. Absorbance was measured and quantified by using Oasys UVM340 microplate reader at 570 nm. The concentration of NO was evaluated based on the nitrate standard curve.

The NO assays were preformed essentially as above but the supernatant from LPS treated RAW264.7 macrophages was used to determine the total amount of NO secreted into the media using the Thermo Scientific Total Nitric Oxide Assay Kit according to the manufacturer’s instructions. RAW264.7 macrophages were seeded in 24-well Corning CellBIND plates and allowed to attach overnight. In the morning the LPS samples were diluted to the indicated concentrations in DMEM and placed on the monolayers. At 6 and 24 h the media was removed and NO measured. This nitric oxide assay measured amount of nitrite (NO₂-) and nitrate (NO₃-) released into the cell culture media. In the assay, the enzyme nitrate reductase converts nitrate to nitrite. Nitrite in each sample supernatant is then detected as a colored azo dye product of the Griess reaction that absorbs visible light at 540 nm. Total nitric oxide (NO) contributed by nitrate and nitrite release in response to LPS treatment was measured as nitrite after converting all nitrate to nitrite. Absorbance values were compared to a Nitrate Standard curve. Experiments were carried out in biological triplicate.