4.11. Reactive Oxygen Species (ROS) Detection With CellROX-Green Reagent Using Flow Cytometry

The ROS detection was performed with CellROX-Green staining followed by flow cytometry analysis as per manufacturer’s recommendation. Briefly, at the end of the experiment CellROX-Green reagent at a final concentration of 2.5 μM was added to the cells and incubated at 37 °C for 30 min in a 5% CO₂ humidified incubator. The cells were then harvested, washed once with phenol red free media, resuspended in PBS, and immediately subjected to flow cytometry on BD Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The fluorescence intensity was detected with a 530/30 nm band-pass emission filter and FL1 detector. The unstained cells gated using forward-scatter (FSC) and side-scatter (SSC) of light were used to exclude small debris and nullify the background fluorescence. The resulting FL1 data were plotted on a histogram. Data was collected by BD Accuri C6 flow cytometer (BD Biosciences) and analyzed with FlowJo v10.0.6 (FlowJo, LLC, Ashland, OR, USA).