A high-throughput, plate reader-based method for the assessment of oxidative stress in suspension mammalian cells using CellROX Green

The measurement of intracellular reactive oxygen species (ROS) levels provides critical insights into cell health and pathophysiology. Here, we present a semi-quantitative, plate reader-based protocol using CellROX Green that can be used for the real-time measurement of general ROS levels of viable suspension mammalian cells. With menadione being the oxidative stress inducer, a cell concentration of 100,000 cells per well, a CellROX Green concentration of 10 µM, and a CellROX Green incubation time of 1 h was shown to provide the most accurate measurement of intracellular ROS levels in viable human monocyte leukemia (THP-1) cells. Unlike plate reader protocols using other general ROS-sensitive fluorogenic probes, this plate reader protocol eliminates the washing step of the unbound probe prior to measuring fluorescence, making it more suitable for suspension cells. Using the presented protocol, the fluorescence of up to 96 samples can be measured in under 1 min, making it ideal for rapid screening applications where moderate to large differences in oxidative stress levels are to be measured.