Dihydroethidium dhe 2 7 dichlorofluorescin diacetate dcfh da

For the C. elegans assay, the detailed method is provided in the methods section of the supplemental material. In all, four to six synchronized L2 larvae were transferred to agar plates containing 10× the MIC of each antibacterial agent. Under this antimicrobial load, the food for the worms (E. coli bacterium cells) will have died (but do not lyse) during the first day of the experiment and, by the end of the experiment, the worms would have run out of food, thus the offspring would not mature. This did not affect our observations over the timescale of our experiment. Four results were recorded for 5 days: (i) number of live worms, (ii) motility (head swing and body-bending frequency per minute), (iii) growth (body length and body width), and (iv) regeneration (generation of offspring, size, number, and motility of them were considered) rates.
Multiple fluorescent sensor probes were employed, including dihydroethidium (DHE), 2′,7′dichlorofluorescin diacetate (DCFH-DA), and naphthalene-2,3-dicarboxal-dehyde (NDA), all obtained from the Invitrogen to detect the O₂^.−, ROS, and glutathione, respectively.

ROS levels lead to oxidative stress, inflammation, and cellular disruption, while reduced glutathione (GSH) is a cellular antioxidant against oxidative stress, and it is essential to cellular protection [104 (link)]. Multiple fluorescent sensor probes were employed including dihydroethidium (DHE), 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and naphthalene-2,3-dicarboxal-dehyde (NDA), all obtained from the Invitrogen (Carlsbad, CA, USA) to detect the O₂⁻, ROS, and glutathione, respectively.
Different time frames and exposure times were examined to find the optimal conditions to see the differences between the test groups. After the treatment of L2 nematodes with 10× MIC concentration of each PBC and antibiotics in NGM plates at 20 °C, the O₂⁻ and ROS levels were measured after 4 days, while glutathione levels were measured after 24 h. The plates were washed, and the liquid was centrifuged (6000× g) 2 times with PBS. The nematodes were exposed to DHE (20 μM), DCF (10 μM), and NDA (20 μM) probes to measure the O₂⁻, ROS, and glutathione, respectively. The nematodes were incubated with probes at 20 °C for 4 h. The liquid was gently washed 2 times with PBS buffer and the animals were examined on a fluorescence microscope (Zeiss axio imager Z1) with identical exposure time (1.5 s). Densitometry and intensity analysis was performed by Fiji software (ImageJ).