Quantification of Intracellular ROS in C. elegans

Intracellular levels of ROS were measured in C. elegans by using 2,7-dichlorofluorescein diacetate (DCF-DA) as previously reported^{54 (link),92 (link)}. CL4176 worms were cultured in NGM (+DMSO 0.05%) plates, and NGM plates supplemented with 50 μg/mL of DS extract. After 26 h of paralysis induction, worms were collected, washed with M9 buffer, and transferred into a microfuge tube. Worms were pelleted by centrifugation at 14,000 rpm for 2 min, sonicated with a Branson Digital Sonifier 450 (Branson Ultrasonics Corporation, CT, USA) at 30% amplitude (4 cycles of 15 s on/15 s off), and total protein concentration was quantified using the Bradford method. Equal amount of protein from five biological replicates (and three technical replicates) were mixed with PBS in a final volume of 200 μL with 50 μM DCF-DA. Fluorescence was acquired in a BioTek Cytation 5 plate reader (Agilent, USA) using excitation at 485 nm and emission at 530 nm.