Durapore 1.2 μm filters

CB₁ receptor binding assays were performed using a Multiscreen Filtration System with Durapore 1.2-μM filters (Millipore, Bedford, MA) as described previously (Hillard et al., 1995 (link)). Incubations (total volume = 0.2 mL) were carried out using TME buffer containing 1 mg/mL bovine serum albumin (TME/BSA). Membranes (10 μg protein per incubate) were added to the wells containing 0.25, 0.5, 1.0, or 2.5 nM ³H-CP 55,940. Ten μM Δ⁹-tetrahydrocannabinol was used to determine non-specific binding. K_D and Bmax values were determined by nonlinear curve fitting to the single site binding equation using GraphPad Prism (San Diego, CA, USA).

Membrane preparations from amygdala tissue from 20 P40 (+/− 2 days) animals (10 male/10 female, 50% LPS/control; 6 litters) were generated as previously described (Lee and Hill, 2013). In brief, tissue samples were homogenized in TME buffer (50mM Tris-HCl, 1mM EDTA, 3mM MgCl₂, pH 7.4), using a Dounce homogenizer, centrifuged at 18,000 × g and the resulting pellet was resuspended in 20 volumes of TME buffer. Protein content was determined using the BCA method using a commercially available kit (Pierce Biotechnology, Rockville, IL, USA). Samples were then aliquoted and stored at −80°C until further testing for measures of CB1 receptor binding and FAAH activity (see below).
CB1 receptor agonist binding parameters were determined using a previously detailed radioligand binding assay (Lee and Hill, 2013) using a Multiscreen Filtration System with Durapore 1.2-μM filters (Millipore, Bedford, MA). In brief, membranes (10μg protein) were added to wells in triplicate containing different concentrations of radioactively labeled CB1 agonist ([³H]CP 55,940). Addition of the CB1 antagonist AM251 (10μM) was used to determine non-specific binding. Dissociation constant (K_D) and maximum binding (B_max) were determined by nonlinear curve fitting of specific binding data to the single site binding equation using GraphPad Prism (GraphPad 7, La Jolla, CA, USA).