Quantifying ROS Levels in C. elegans

The ROS level in C. elegans was determined using 2′,7′-dichlorofluorescin diacetate (DCFH-DA) (Sigma, St. Louis, MO, USA) as previously described [17 (link)]. Briefly, approximately 1000 nematodes treated with 6-OHDA and astragalan were collected, washed, and homogenized in 350 μL of PBS (50 mM, pH 7.8) with 0.1% Tween 20 on ice. The supernatant was collected by centrifugation, and its protein content was determined by BCA assay kit (Thermo Fisher, Waltham, MA, USA). Then 50 μL of the supernatant was transferred into a black 96-well plate and incubated with 50 μL of 100 μM DCFH-DA. The DCF fluorescence was determined in a Fluoroskan Ascent FL microplate reader (Thermo, Waltham, MA, USA) every 10 min for 2 h at an excitation of 485 nm and an emission of 535 nm. The ROS level was calculated as the DCF fluorescence per μg proteins.