Dcfh da ros assay kit

Cell intracellular ROS was measured using DCFH-DA ROS Assay Kit according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). Briefly, HUVECs (3 × 10⁵ per well) were seeded in 6-well plates and were treated with compounds for 1 hour. LPS was then added to cells and incubated for 24 hours. For the assessment of ROS, after wash the cells with PBS, DCFH-DA (10 μM) was added into the cells and incubated for 30 min at 37 °C. Cells were collected and free DCFH-DA was removed. The ROS level was examined using a flow cytometer (BD FACS Aria, Franklin Lakes, NJ, USA).
For determination of ROS production in the lung, LPS-induced ALI mice models were treated with the indicated drugs for 24 hours. Lung samples were collected, ground in culture medium and isolated using a 40 μm cell strainer (Corning, Corning, NY, USA). Samples were rinsed with PBS three times and incubated with DCFH-DA at 37 °C. ROS level were analyzed using a flow cytometer.