Oxidative Stress Measurement in Nematodes

The nematodes were rinsed with M9 buffer solution to remove OP50 after a 5-day treatment with 0.18 or 0 mg/mL of APPA. Afterward, 30 nematodes were treated with 100 µM of 2, 7-dichlorofluorescein diacetate (H₂DCF-DA) (Meilunbio, Shanghai, China), incubated at 35 °C for 30 min, anesthetized with levamisole, and then placed on 2% agarose [52 (link)]. Subsequently, 30 nematodes per group were measured and detected by blue fluorescence (Ex 430–455 nm) under the Olympus X71 fluorescent microscope (Tokyo, Japan), viewed through a ten-fold objective. The nematode size was determined from the bright field image. Finally, their relative fluorescence intensity was determined by the integrated density and quantitative fluorescence intensity by ImageJ 15.2v [63 (link)]. Then, the fluorescence intensity of nematodes per unit area was calculated. The data were analyzed in terms of the fluorescence intensity of the experimental group relative to the blank group.