Sr144528

Cells in normal growing media were seeded into a 96-well plates at a concentration of 10,000 cells per well in triplicate. The following day, cells were treated with different extracts/fractions/compounds, or media/solvents alone for controls. In experiments where CB₁ antagonist AM251 (Tocris, 1117/1) or CB₂ inverse agonist SR144528 (Abcam, ab146185) were used, cells were treated with the antagonist/inverse agonist 1 h prior to treatments. Viability was quantified using the XTT viability assay (Biological Industries). Cells were incubated in medium for 48 h, then XTT reagent (2,3,-bis (2-methoxy- 4-nitro- 5-sulfophenyl)- 5-[(phenylamino)- carbonyl]-2H- tetrazolium inner salt) was added for 2 h at 37°C in a humidified 5% CO₂–95% air atmosphere. Absorbance was recorded by a Synergy H1 hybrid reader photometer (BioTek) at 490 nm with 650 nm of reference wavelength. Cell survival (% viability) was estimated using the equation: % cell survival= (A₄₉₀-A₆₅₀) of treatment / (A₄₉₀-A₆₅₀) of solvent control × 100. A₄₉₀ and A₆₅₀ are the absorbencies of the XTT colorimetric reaction. Absorbance of medium alone (blank) was also reduced from specific readings.

Restek phytocannabinoid standards were used in this study: ∆-9 tetrahydrocannabinol (Δ-9 THC, 34067), cannabichromene (CBC, 34092), cannabidiol (CBD, 34011) at a concentration of 1 mg/mL, originally dissolved in methanol. For quantification of phytocannabinoids the standards were dissolved in methanol at various concentrations from 0–25 μg/mL. Mitomycin-C (MMC) was dissolved in water at a stock concentration of 800 μg/mL. Inverse agonists (IA) CB1 (AM251, Abcam ab120088), CB2 (SR144528, Abcam ab146185), TRPA1 blocker (HC-030031, Abcam ab120554) and TRPV1 and TRPV2 antagonists (Abcam ab141772 and Tranilast 1098/10, respectively; Tranilast is a TRPV2 inhibitor [40 (link)]) were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10mM.

SR144528—a CB2 receptor antagonist, was purchased from Abcam, Cambridge, UK. A967079—a TRPA1 Receptor antagonist and BCTC—a TRPV1 Receptor antagonist, were purchased from Alomone Labs, Jerusalem, Israel. GSK2193874—a TRPV4 antagonist, was purchased from SIGMA-ALDRICH, Rehovot, Israel. CID16020046—a GPR55 antagonist, was purchased from Cayman, MI, USA and GW9662—a PPARγ antagonist, was purchased from Enzo Life Sciences, New York, NY, USA.