Reactive oxygen species (ROS) measurement

Total levels of reactive oxygen species were measured using 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent dye to determine the generation of free radicals during RMT on UPEC along with other controls⁵⁹. RMT exposed, RF treated, and RF-untreated control cells at 25 °C and 46 °C with or without MCSNPs samples were washed three times in PBS and equal cell numbers of all samples including control were adjusted to an OD₆₀₀ equivalent of 0.1. The bacterial samples were stained with 10 μM ROS-specific fluorescent dye DCFH-DA for 30 min in the dark at the room temperature and were washed with PBS once. Fluorescence was measured using a microplate reader with excitation at 488 nm and emission at 525 nm (BioTek, USA). The total ROS level is presented as a percent relative to the control at room temperature with or without MCSNPs. All the samples were measured in triplicate and data is presented as mean ± SD.