2.8. Live/dead staining and ROS detection assays
The bacterial viability was investigated using Live/Dead Bacterial Viability Kit. K.pneumoniae after different treatments was collected by centrifuging (8000 rpm, 5 min) and then co-dyed with Calcein-AM and propidium iodide (PI) in dark for 30 min. After that, the bacteria were washed with normal saline to remove excess dye. Finally, bacterial solutions were dropped on the glass slides and imaged Confocal Microscope (FV1200, Olympus).
For the analysis of intracellular ROS content, 108 CFU mL-1 K.pneumoniae cells were added into the PBS (control), HMPB (100 μg/mL), ofloxacin (10 μg/mL), and OHH NPs (100 μg/mL) and further incubated for 30 min, the net concentration of the HMPB substrate in all sample groups has been maintained at 100 μg/mL. After that, the K.pneumoniae cells were rinsed with medium and stained with DCFH-DA under 37 °C for 20 min and then analyzed using a confocal microscope (FV1200, Olympus). Quantitative determination of the ROS content in the cell samples was further carried out as a complement to the characterizations above.