4.4. Measurement of ROS Production

ROS were quantified using 2′,7′-dichlorofluorescin-diacetate (H₂-DCF-DA, Sigma-Aldrich, St. Louis, MO, USA), as previously described [17]. Upon cleavage of the acetate groups by intracellular esterase and oxidation, the H₂−DCF-DA is converted to the fluorescent 2′,7′-dichlorofluorescein (DCF). Briefly, the intestinal cells were seeded into 96-well plates and allowed to adhere overnight. ROS level was measured after the exposure to BOS (1 µg/mL) or to CUR (1 µg/mL) for 24 h. Treatments were removed and H₂DCF-DA was added to obtain a final concentration of 50 µM in each well. The plate was incubated for 30 min at 37 °C and washed with phosphate-buffered saline (PBS). DCF fluorescence intensity was measured at excitation 485 nm–emission 535 nm, using VICTOR^TMX3 Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA), before and after the addition of 500 µM H₂O₂ on each well.