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.