Electrophorus electricus electric eel

After the experiments were completed, mouse brains were isolated and sections of the prefrontal cortex, hippocampus, and brainstem were collected. Frozen brain tissues were homogenized in lysis buffer (50mM TRIS-HCl solution with 150 mM NaCl, 1% Triton X-100, pH = 8) using an ultrasound homogenizer. Additionally, 20 mg tissue/mL of buffer was used. The prepared homogenates were centrifuged at 14000 rpm for 10 min at 4 °C. Then, the supernatant was used for AChE activity determination. The AChE activity in examined samples was measured quantitatively via Ellman’s method [52 (link)], with modifications, using a 96-well microplate reader (Epoch. BioTek, Winooski, VT, USA). The mixture was prepared by mixing 0.4 mL supernatant with 2.6 mL of DTNB solution (10 mM 5.5 dithiobis 2-nitrobenzoic acid in phosphate buffer). Next, 155 µL of the mixture was transferred to a 96-well plate in triplicate for each sample. After that, the plate was incubated for 10 min at 37 °C. After incubation, 10 µL of ACTI (7.5 mM acetylthiocholine iodide in distilled water) was added to each well, and absorbance was measured at 412 nm for 10 min at 1 min intervals. AChE activity was determined using a calibration curve performed at each measurement using acetylcholinesterase from Electrophorus electricus (electric eel, Sigma, Poznań, Poland) and expressed in mU min⁻¹ mg⁻¹.