Nagly

The following native and deuterated standards were purchased from Cayman Chemicals (Ann Arbor, MI, USA): 2-AG, AEA, PEA, SEA, OEA, LEA, POEA, EPEA, DEA, NA-Gly, 2-AG-d₈, AEA-d₈, PEA-d₄, SEA-d₃, OEA-d₄, 12-[[(cyclohexylamino)carbonyl]amino]-dodecanoic acid (CUDA). Acetonitrile and methanol were from Merck (Darmstadt, Germany). Isopropanol was from VWR PROLABO (Fontenay-sous-Bois, France). Acetic acid was purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI, USA). Butylhydroxytoluene (BHT) was from Cayman Chemical (Ann Arbor, MI, USA) and ethylenediaminetetraacetic acid (EDTA) from Fluka Analytical, Sigma-Aldrich (Buchs, Switzerland). Glycerol was from Fischer Scientific (Loughborough, UK). All solvents and chemicals were of HPLC grade or higher. Water was purified by a Milli-Q Gradient system (Millipore, Milford, MA, USA).

AEA, O-1602 and AbnCBD were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). NAGly was bought from both Cayman and Enzo Biosciences (in order to exclude the possibility that lack of responses were related to a specific company’s synthesis/production; results were equivalent for both suppliers). Forskolin, CP55, 940, SR141716A, AM251 and AM630 were purchased from Tocris Bioscience (Bristol, UK). Phorbol-12-myristate-13 acetate (PMA) was purchased from Sigma Aldrich (St Louis, MO, USA). U0126 was purchased from Cell Signaling Technology (Danvers, MA, USA). All cannabinoid and lipid ligands were diluted in ethanol prior to storage in single use aliquots at −80 °C. The lipophilic properties of cannabinoids and lipid ligands such as NAGly cause them to adsorb onto the hydrophobic surfaces of plasticware and interact with serum components. To sustain drug availability in assays, the vessels in which cannabinoids were diluted were silanised (Coatasil, ThermoFisher AJA2293; Waltham MA, USA) and autoclaved prior to use, and assay media or HBSS were supplemented with 1 mg/ml bovine serum albumin (BSA, MP Biomedicals ABRE, Santa Ana, CA, USA).