Fluorometric intracellular ros assay kit

Detection of intracellular ROS in DEFs was performed with a fluorometric intracellular ROS assay kit (Sigma–Aldrich, USA) based on a superoxide fluorescent probe (dihydroethidium). DEFs were plated in 96-well black microplates with clear flat bottoms to a cellular concentration of 1 × 10⁴ cells/mL. The cells were then allowed to adhere for 24 h and infected with DTMUV, and the cells were harvested according to the instructions at 6, 12, 24, 36, 48 and 60 h of infection. The ROS reagent was reconstituted with dimethyl sulfoxide and then diluted in the assay buffer. Shortly afterwards, the ROS reagent solution was added to each well, and the plates were incubated for 1 h. Subsequently, the data were collected using a fluorescence microscope, and the fluorescence intensity was measured in a multifunctional microplate reader (Thermo Scientific, Massachusetts, USA) with an excitation wavelength of 520 nm and an emission wavelength of 605 nm. The data are shown as relative fluorescence compared to the fluorescence of the negative control.

ROS (superoxide and hydroxyl radicals) were detected with the Fluorometric Intracellular ROS Assay Kit (Sigma-Aldrich, USA) following the manufacturer’s protocol using fluorometric sensors localized to the cytoplasm, resulting in a fluorometric product proportional to the amount of ROS present. The fluorescence intensity was determined at λex = 540 nm and λem = 570 nm. Fold increases in ROS levels were determined by comparison with the control group.