Am630

In both cell types, cells were treated with (0.1 µM) PEA dissolved in FM-LipoMatrix^® (a new technology based on solvent patent N°102017000036744 of noiVita srls, produced by Pro-Bio INTEGRA srl, Rovigo, Italy) in the presence or absence with lipoic acid (50 µM) plus vitamin D3 (100 nM) prepared directly into medium of stimulation (named LA + vitD) [39 (link)]. To verify the effectiveness of this new product the effects were compared to the classic commercial product (0.1 µM micronized PEA) and the solvent technology was also tested alone. The concentration of PEA was obtained by literature to maintain the antioxidant effect [53 (link)] and confirmed by dose-response study (data not shown) [54 (link)]. To verify the mechanism involved, some experiments were carried out also in presence of 30 min pre-treatments with both 10 µM AM251 and 10 µM AM630 (Cayman Chemical Company, Ann Arbor, MI, USA) [55 (link)], the specific CB1 and CB2 inhibitors, respectively. Finally, Lipopolysaccharides (LPS, Sigma-Aldrich, Milan, Italy) 500 ng/mL was used to verify the neuroprotective properties, pre-treating the cell for 24 h [56 (link)].

AM251, SR141716A (Rimonabant), URB447, LH21, AM630, O-2545, and CP55,940 were obtained from Cayman Chemical (Ann Arbor, MI) and Gp1a was obtained from Tocris Bioscience (Minneapolis, MN).

Jurkat, BV2 and RAW264.7 cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ in RPMI supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine and 1% (v/v) penicillin/streptomycin (complete medium). VCE-003 was synthesized as previously described [20] (link), while AM630, T0070907 and WIN-55,212-2 were obtained from Cayman Chem (Ann Arbor, MI, USA), and JWH-133 was purchased from Tocris Bioscience (Bristol, UK). All other reagents were from Sigma Co (St Louis, MO, USA).

Recombinant human IL-6 was purchased from GenScript (Piscataway, NJ). Cannabinoid CB2 inverse agonist SR144528 and CB1 antagonist SR141716 were provided by NIH-NIDA-NDSP program. AM630 (CB2 inverse agonist) and HU308 (CB2 agonist) were purchased from Cayman Chemical (Ann Arbor, MI). Human IgM ELISA Kit (Immunology Consultants Laboratory, Inc., Portland, OR) was used according to manufacturer’s recommendations. ³H-Thymidine (46.5 Ci/mmol) was purchased from PerkinElmer (Boston, MA). TPA and LPS were from Sigma-Aldrich (Saint Louis, MO).
The human SKW 6.4 cell line was obtained from American Type Culture Collection. Cells were cultured in RPMI-1640 medium with 10% heat-inactivated fetal bovine serum (FBS), 2 mmol/L glutamine, and 100 U/mL penicillin–streptomycin (Sigma-Aldrich) at 37°C with 5% CO₂. For the experiments, SKW 6.4 cells (5 × 10⁴/ml) were placed in 96-well flat-bottomed microtiter plates (200 μl/well) and were incubated with or without IL-6, SR144528 and the other agents for various times at 37°C. CB2 ligands were prepared in dimethyl sulfoxide (DMSO) stock solutions of 50 mM and diluted by medium before application. For all the cell cultures with CB2 ligands, the final concentrations of DMSO were always equal or less than 0.05%. Control cells were also treated with an equivalent amount of DMSO without the drugs.

The 6-OHDA was purchased from Sigma-Aldrich (St. Louis, MO, USA). D-amphetamine was provided by the NIDA Drug Supply Program. GW842166X and AM630 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). GW842166X and AM630 were first dissolved in DMSO (Sigma-Aldrich) and then mixed with TWEEN-80 (Sigma-Aldrich). Then, sterile saline was added to the mix solution to create a final working solution with 2.5% DMSO + 2.5% TWEEN-80 + 95% saline. Picrotoxin and all other common chemicals were obtained from Sigma-Aldrich. 6-Cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX) and D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5) were obtained from Tocris Bioscience (Ellisville, MO, USA).