Planarian Acetylcholinesterase Activity Assay

After exposure, the planarians were washed 3X with IO water and then homogenized in 200 µL 1% Triton X-100 in PBS as described in (Hagstrom et al, 2017 (link); 2018 (link)). Briefly, after sitting on ice for about 15–20 min, the homogenate was centrifuged at 20,817 x g at 4°C for 30 min. The supernatant (clarified homogenate) was transferred to a clean, chilled tube and subsequently used. An Ellman assay (Ellman et al, 1961 (link)) was performed on the clarified homogenate using an Acetylcholinesterase Activity Assay kit (Sigma-Aldrich). Absorbance was read at 412 nm every minute for 10 min using a SpectraMax ABS Plus (Molecular Devices, San Jose, CA) spectrophotometer. AChE activity was calculated as the rate of change of absorbance per minute during the linear portion of the reaction. AChE activity was normalized by protein concentration as determined by a Coomassie (Bradford) protein assay kit (Thermo Scientific, Waltham, MA) and compared to the average respective solvent control samples (set at 100% activity) tested on the same day. Activity measurements were performed with 3 technical replicates per condition and 4 independent experiments (biological replicates).