Xmark reader

The Ca9-22 cells were seeded in the 6-well culture plates and exposed to various concentrations of PAC (2.5, and 5 μM) and cisplatin (0, 0.01, 0.1, 0.5, 0.8, and 1 nM) for two weeks. After the completion of the culture period, the culture medium was aspirated, and the adherent cells were washed with PBS. Subsequently, the cells were fixed with 4% methanol at room temperature for 10 min and then stained using 0.05% crystal violet. Following a 15 min incubation at room temperature, the dye was carefully removed, and the wells were thoroughly washed with PBS. The plates were allowed to dry, and colony formation was observed and captured using an inverted microscope. To quantify the colony formation, the crystal violet was solubilized with 200 µL of a 30% (v/v) acetic acid solution. The absorbance was measured at 570 nm using a Bio-Rad xMark reader in a 96-well plate (Bio-Rad, Mississauga, ON, Canada).

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.