Quantifying Cellular Oxidative Stress

Levels of intracellular ROS and NO and extent of lipid peroxidation in primary murine keratinocytes were determined using H₂DCF-DA (Invitrogen/Life Technologies), DAF-FM-DA (Sigma), or C11-BODIPY^581/591 assays (Invitrogen/Life Technologies), respectively. H₂DCF-DA allows detection of intracellular H₂O₂, but it also detects oxygen radicals [52 (link)]. DAF-FM-DA is a probe to detect NO, but it only works under aerobic conditions and it is likely to react with an oxidative product of NO, rather than with NO itself [53 (link)]. Cells were incubated for 30 min with 50 μM H₂DCF-DA or 5 μM DAF-FM-DA, or for 2 h with 2 μM C11-BODIPY^581/591 in cell culture medium at 37°C prior to detachment by trypsin. Fluorescence was directly measured by flow cytometry using the BD Accuri C6 (BD Biosciences, San Jose, CA).