4.5. Cell Viability Assay

Cell viability was assessed using the thiazolyl blue tetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates, at a density of 1 × 10⁴/well, and incubated at 37 °C in 5% CO₂. Cells were incubated with DMSO (0.3%) (vehicle control) or treated with FAA1 and FAA2, diluted to different concentrations, for specified periods of time. For the analysis of the effects of cannabinoid receptor, cells were incubated with the CB1 antagonist, 10 μM LY3210135, and/or the CB2 antagonist, AM630, 30 min before the treatment with fatty acid amides for 12 h; 120 µg/mL FAA1 and FAA2. After incubation, the medium was removed and replaced with serum-free medium containing 0.5 mg/mL MTT and incubated for 3 h at 37 °C. The MTT reagent was removed, the formazan crystals were solubilized using DMSO and the absorbance was recorded at 570 nm using a microplate spectrophotometer (Biorad Model 450 Microplate Reader, Hercules, CA, USA). Untreated cells were used as control.