2.11. Determination of Reactive Oxygen Species (ROS) by the DCFH-DA Assay

A fluorescent 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay was performed to determine the intracellular ROS concentrations according to the method reported by Miranda-Rottmann [28] with modifications. Cells were seeded at 1 × 10⁵ cells/well in 96-well plates in a final volume of 100 µL of culture medium per well. When the cells reached a confluence of 80%, HT-29 and RAW 264.7 cells were incubated with H₂O₂ and LPS, respectively in the presence or absence of Ach and internal control (NAC and 5-ASA) for 1 h with 5% CO₂ at 37 °C. After removing the medium, cells were washed twice with 50 µL/well of PBS, incubated at 37 °C for 30 min with 25 μM DCFH-DA, and stored under at −20 °C. For the DCFH-DA assay, a culture medium was used without phenol red and without supplementation to avoid interference with fluorescence emission. The fluorescence was measured at Ex/Em: 485/530 nm using a fluorescence microplate reader.