Quantification of Nitric Oxide Secretion

The level of NO secreted into the culture medium was determined by the Griess reaction as previously described [38 (link)]. Equal volumes of supernatant, Griess reagent A [0.1% N-(1-naphthyl)ethylenediamine dihydrochloride] and Griess reagent B (1% sulfanilamide in 5% phosphoric acid) (both from Sigma-Aldrich, St. Louis, MO, USA) were mixed, and the absorbance of the prepared solution was measured immediately at a wavelength of 540 nm in a Tecan Infinite 200 Pro spectrophotometer (Tecan, Mannedorf, Germany). The data were normalized to the level of NO released from the vehicle group in control OCCs (Experiment 1) or the vehicle group from the corresponding type of OCCs (Experiment 2) and are presented as the percentage of the control ± SEM.