2.10.1. Measurement of ROS Generated by RAW 264.7 Cells

The intracellular ROS generated by RAW 264.7 cells was determined using a 2′,7′-dichlorofluorescin diacetate (DCFH-DA) assay. The DCFH-DA enters the cell where it reacts with ROS to form a highly fluorescent compound of dichlorofluorescein (DCF). Briefly, RAW 264.7 cells (2 × 10⁵ cells/well) were seeded onto a 6-well plate and allowed to attach for 24 h before treatment. The culturing medium was then replaced with the release medium from each ulvan/chitosan complex film and added with LPS (1 μg/mL); the cell culture was continued for 1 more day. Finally, the medium was removed, and a fluorescent dye (20 μM DCFH-DA) was added into the well for a 30 min incubation. After washing cells with PBS, the cells were observed by inverted fluorescence microscope (CKX41, Olympus, Tokyo, Japan) and analyzed by flow cytometry (BD FACSAria, BD Biosciences, San Jose, CA, USA).