With reference to Song et al (6 (link)), CL4176 nematodes were synchronized at 16˚C for 48 h, cultured at 25˚C for 40 h then rinsed twice with M9 buffer to remove E. coli. The nematodes were then homogenized and protein abundance was measured by the bicinchoninic acid assay (Beyotime Institute of Biotechnology). The levels of MDA, SOD and CAT were determined using Total Superoxide Dismutase Assay (Beyotime Institute of Biotechnology), Catalase Assay (Beyotime Institute of Biotechnology) and Malondialdehyde Detection (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. ROS accumulation was determined with reference to Wang et al (29 (link)). Briefly, 50 µl of nematode supernatant was added to each well of a 96-well plate and 50 µl of 100 µM DCFH-DA solution (a fluorescent probe) (Beyotime Institute of Biotechnology) was added to give a final DCFH-DA concentration of 50 µM, The solutions were thoroughly mixed by shaking for 30 sec. Fluorescence detection was performed using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Detection was conducted once every 10 min, and ROS changes within 100 min were counted.