Oxidative stress analysis in BPAE and Raw cells using CellROX® Deep Red Reagent (Cat. No. C10422)

Bovine pulmonary artery endothelial (BPAE) cells or RAW macrophage cells were plated in 96 well plates. BPAE cells were treated with or without 100 µM menadione for 1 hr. 100 µM of superoxide scavenger, MnTBAP was added to some of the control and menadione-treated wells for the last 30 mins of incubation. RAW cells were treated with or without 500 ng/ml of LPS +/- 150 nM diphenyleneiodonium (DPI), a NADPH oxidase inhibitor. The cells were then stained with 5 µM Far Red ROS Sensor by adding the probe to the complete media. The cells were then washed with PBS and analyzed on a Thermo Fisher Cellomics ArrayScan® VTI. MnTBAP or DPI treatment inhibited ROS caused by menadione or LPS, respectively, confirming that the signal was due to ROS induced by these compounds. ***a The values are significantly different from controls with P = 0.0001 The values are significantly different from controls with P = 0.0001; ***b values were signficantly different from drug treated cells with P = 0.0001