ROS Generation Measurement Using DCFDA

ROS generation was measured according to the previously described method using 2′,7′-dichlorofluorescein diacetate (DCFDA) [52 ,95 (link)]. Bacterial cells (10⁶ CFU/mL) were treated with or without AgNPs at the required temperature for 12 h. After incubation, cells were centrifuged at 4 °C for 30 min at 300× g, after which the supernatant was treated with 100 μM DCFDA for 1 h. The amount of ROS produced in the sample was detected at the excitation wavelength of 485/20 nm of fluorescence excitation and emission wavelength of 528/20 nm using a fluorescence multi-detection reader (BIOTEK, Winooski, VT, USA).