Evaluating ROS Production in IEC-6 Cells

The probe 2′,7′-dichlorofluorescein-diacetate (H₂DCF-DA) was used to evaluate ROS intracellular production [26 (link)]. IEC-6 cells were plated in 24-well plates (8.0 × 10⁴ cells/well). After adhesion, almond ASE and CD-ASE (50–5 μg/mL) were added for 1 h alone and then simultaneously to LPS (10 μg/mL) plus IFN (10 U/mL) for 24 h. In other experiments, the cells were treated with the tested products, at the same concentrations, alone for 1 h and then simultaneously to H₂O₂ (1 mM) for another 1 h. IEC-6 cells were then collected, washed with PBS and incubated in PBS plus H₂DCF-DA (10 μM). After an incubation of 15 min at 37 °C, cell fluorescence was evaluated using a fluorescence-activated cell sorter (FACSscan; Becton Dickinson, Franklin Lakes, NJ, USA), and was analyzed by Cell Quest software version 4 (Becton Dickinson, Milan, Italy) [27 (link)].