4.3. Fatty Acid Amide Hydrolase Assay

FAAH enzymatic activity was studied by using membranes prepared from BV-2 cells treated with vehicle or pre-treated with 5 μM URB597 for 4 h and incubated with 30 μM Aβ25-35 for 24 h. Membrane preparations were then incubated with [¹⁴C]-AEA (85.0 mCi/mmol, ARC St. Louis, MO, USA) properly diluted with AEA (Cayman Chemicals, Ann Arbor, MI, USA) in 50 mM Tris-HCl, pH = 9, for 30 min at 37 °C. The [¹⁴C]-Ethanolamine produced from [¹⁴C]-AEA hydrolysis was measured by scintillation counting of the aqueous phase after the extraction of the incubation mixture, with two volumes of CHCl₃/CH₃OH (1:1, v/v); the activity was expressed as percentage of the maximum effect observed in the absence of treatments.