Equine serum bche

The AChE and BChE inhibitory activities of compounds were determined by using a slightly modified Ellman’s method [30 (link),31 (link)]. Electric eel AChE, equine serum BChE, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), phosphate buffer solution (PBS, pH 8.0), acetylthiocholine (ATC) iodide, and butyrylthiocholine (BUC) iodide were purchased from Sigma-Aldrich (Steinheim, Germany). Tacrine was used as positive control. Enzyme solutions were prepared at 2.0 U/mL in 2-mL aliquots. The assay medium consisted of 10 μL of enzyme, 40 μL of PBS, 20 μL of 0.01 M DTNB, and 10 μL of tested compound. Assayed solutions of tested compounds were pre-incubated with corresponding ChE for 5 min. The reaction was initiated by the addition of 20 μL of 0.01 M substrate (ATC or BUC). The activity was determined by measuring the increase in absorbance at 410 nm at 37 °C in 2-min intervals using Tecan Spark multimode microplate reader (Mannedorf, Switzerland). The percentage of inhibition (I) was calculated from the measured data as follows: I = (A_c − A_i)/A_c × 100%, where A_i and A_c represent the change in the absorbance in the presence of an inhibitor and without an inhibitor, respectively.

l-Glutamine, fetal bovine serum, penicillin, streptomycin, amphotericin B, HEPES, Hanks’ salts solution, trypsin, DMEM, (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from PanEco, Moscow, Russia. HEPES, KCl, CaCl₂, MgCl₂, DMSO, Triton X-100, d-glucose, non-essential amino acids, Hoechst 33258, human erythrocyte AChE, equine serum BChE, acetylthiocholine iodide, CHAPS, EDTA, dithiothreitol, PNU 120596, protease inhibitor cocktail, SCP0139, and 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) were from Sigma-Aldrich, St. Louis, MO USA. Arachidonic acid and Z-VAD-FMK were purchased from Cayman Europe, Hamburg, Germany. Ac-DEVD-AFC and Ac-LEHD-AFC were from Tocris Bioscience, Bristol, UK. Fluo-4AM and probenecid were from Thermo Fisher Scientific, Waltham, MA, USA.