Cannabinoid Mixture Evaluation on Cell Proliferation

Cell proliferation was evaluated using classical MTT assay and confirmed by nucleus staining with Hoechst. As previously described [28 (link),29 (link)], 10⁵ cells/well from Ca9-22 and GMSM-K cells were seeded in 12-well plates, cultured overnight and then exposed for 24 h to CM to concentrations (from 0.1 $\mathsf{\mu }\mathrm{g}\mathsf{/}\mathrm{m}\mathrm{L}$ to 2 $\mathsf{\mu }\mathrm{g}\mathrm{/}\mathrm{m}\mathrm{L}$ ). After cannabinoid exposure, 1/10 dilutions of 5 mg/mL MTT reagent (MTT; Sigma-Aldrich, Oakville, Ontario, Canada) were added, and cells were incubated for 3 h at 37 °C in the dark. Formazan crystals were solubilized using 1 mL of a 0.05 N HCl-isopropanol solution. Next, 4 × 200 µL of lysis buffer was transferred to a 96-well microplate to measure absorbance at 550 nm by an xMark reader (Bio-Rad, Mississauga, ON, Canada). Percentage of proliferation in living cells was determined by using the following formula: % of cell viability = [(OD_{550 nm} (treated cells) − OD (blank))/(OD (control cell) − OD (blank))] × 100. The IC50 of CM was obtained by plotting the percentage inhibition of cell proliferation against the concentration of the cannabinoid mixture.