2.7. Measurement of reactive oxygen species generation

Reactive oxygen species (ROS) generation was determined by utilizing an oxidation-dependent fluorescence dye with 6-carboxy-2′,7′-dichlorodihydroxy fluorescein diacetate (DCFDA). After 30 min incubation with DCFDA (2 μg/ml) in a 37°C 5% CO₂ incubator, cells were washed twice with PBS. The ROS generation as green fluorescence was measured at excitation and emission wavelengths of 485 nm and 535 nm respectively by a Synergy HT Microplate Reader (Biotek, VT, USA), fluorescence imaging by Sapphire Biomolecular Imager (Azure Biosystems, USA), and flow cytometry (BD FACSCanto, BD biosciences, USA). Quantification of images was done using AzureSpot (Azure Biosystems, USA) and flow cytometry data was analyzed using the FlowJo v10 software.