Cellular reactive oxygen species detection kit

RAECs were cultured in 96‐well black‐wall/clear‐bottomed microplates and plate cells overnight in growth medium at 10,000–40,000 cells/100 μl. Cellular reactive oxygen species were measured according to the assay protocol (Cellular Reactive Oxygen Species Detection Kit; Abcam, USA). 100 μl/well of ROS Red working solution was added to the cell plate and incubated in 5% CO₂ at 37°C for 1 hr. Cells were treated with 20 μl of 11‐fold test compounds in PBS for 30 min., 2, 4 hrs. For control wells (untreated cells), equivalent amount of compound buffer was added. The fluorescence increase at Ex/Em = 520/605 nm was monitored with bottom read mode.

Vero cells (1 × 10⁶ cells/well) seeded in 6-well plate were infected and treated as described above. The ROS level was detected using a Cellular Reactive Oxygen Species Detection Kit (Abcam, UK) according to user manual. The relative fluorescence units (RFU) was monitored at Ex/Em = 540/570 nm.

Intracellular ROS was detected using the cellular reactive oxygen species detection kit as per the manufacturer’s instructions (Abcam, Tokyo, Japan). The cells were treated with or without citric acid for 2 h, washed twice with washing buffer, and treated with 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) for 1 h. The cells were washed again with washing buffer to remove DCFDA, and fluorescence (excitation/emission = 495/529 nm) was measured using a microplate reader.