Cytation 5 plate reader

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.

Low-profile PCR tubes (200 μL capacity) were first blocked with 150 μL PBS+0.2% BSA at room temperature for 1 h and then washed twice with DPBS (Fisher, Cat# SH30028FS). Purified Stx2a-A1 (37.5 μM in DPBS) was incubated with an equal volume of DARPin (150 μM in DPBS) or DPBS alone at 37 °C for 20 min to allow DARPin to form complexes with the toxin. Next, 1.6 μL of this mixture was added to 1× NEBExpress Cell-free E. coli Protein Synthesis System (NEB, Cat# E5360S) containing 3 μL of S30 extract, 6 μL of Protein Synthesis Buffer, 0.2 μL T7 RNA polymerase, and 0.2 μL RNase Inhibitor, Murine. The reaction was incubated at 37 °C for 30 min, during which time the toxin Stx2a-A1 is able to inactivate the ribosomes. Next, a plasmid DNA encoding a reporter eGFP gene under a T7 promoter (75 ng) was added to the reaction to initiate RNA synthesis and protein translation. The reaction was carried out at 37 °C for 6 h, followed by overnight incubation at 4 °C to ensure complete folding of eGFP. A total of 45 μL of ddH₂O was then added to each tube, and the mixture was transferred to a black 384-well plate (25 μL/well) for the quantification of fluorescence intensity using a Cytation 5 plate reader (Agilent). The positive controls contained no toxin, and the negative controls contained toxin, but no DARPin.