Neuroprotective Effects of Cannabinol

The SH-SY5Y cell line was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells were cultured using Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F12) medium (Sigma-Aldrich, Saint Louis, MO, USA), containing 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, Saint Louis, MO, USA), 1% glutamine, and 1% penicillin-streptomycin. In order to induce neuronal differentiation, SH-SY5Y cells were treated with 10 µM RA for 5 days. At the end of the 5-day-differentiation, the medium was replaced with fresh medium with or without CBN at the concentration 10 or 20 µM (dissolved in DMSO; final DMSO concentration <0.1%). A previous report showed no cytotoxicity at these concentrations [12 (link)]. After 24 h of pre-treatment, the medium was changed with fresh medium with 1 mM MPP⁺ dissolved in water for 48 h. The MPP⁺ concentration and time of exposure were chosen on the basis of a previous report [15 (link)]. Control cells were incubated with DMEM/F12 medium supplemented with 10% FBS.