Quantifying Oxidative Stress by Nitrotyrosine

To assess if the test compounds induce oxidative damage, cells were seeded, treated, fixed, and permeabilized as described above. Shikonin was used as a positive control [86 (link)]. After blocking, cells were exposed to mouse monoclonal anti-3-nitrotyrosine antibody (1:500 in blocking buffer, 15 µL, overnight). After exposure to goat anti-mouse Alexa Fluor 488 secondary antibody (1:10,000, 15 µL, 1 h), cells were stained using DAPI and stored in PBS (Figure S2), images were aquired using an INCell 2200 analyzer (10× magnification) and analyzed using IN Carta image analysis software as described above (GE Healthcare, Rydalmere, NSW, Australia). Average cellular nitrotyrosine intensity was automatically quantified for each acquired images. Data were standardized over the non-treated control (100%) and expressed as mean ± SD of at least 8 replicates from one assay. At least 2 × 10³ cells were analyzed for each treatment.