CellROX Assay for Oxidative Stress Detection Using Flow Cytometry

Following manufacturer’s protocol,¹¹¹ CellROX reagent was used to determine ROS from compound exposure. Briefly, MDA-MB-231 or BT-474 cells were seeded at 200,000 cells/mL in a 96-well plate (100 μL of cell suspension per well). After allowing cell attachment, cells were dosed at their respective IC₅₀ values and incubated for 24 h. The following day, the CellROX reagent was added directly to the medium in each well at a final concentration of 5 μM, and cells were again incubated at 37 °C with 5% CO₂ and 95% RH for 30 min. After incubation, the medium was aspirated, and the cells were washed with PBS once, trypsinized, and centrifuged. Cell pellets were washed in PBS once more and resuspended in 100 μL of PBS. The cell suspension was exposed to 1 μg/mL propidium iodide to detect cell lysis and allowed to incubate at RT for 15 min with a lack of light exposure. Cells were resuspended in FACS tubes at 500 μL and placed on ice until analysis. Data collection was completed using an LSR II flow cytometer, and data analysis was completed using FlowJo, version 10.8.