4.3. Determination of Cell Viability and Neuroprotection Potential by MTT Assay

Cell viability was assessed using the colorimetric MTT assay. SH-SY5Y (differentiated and undifferentiated) and RAW264.7 cells were seeded in 96-well dishes and left overnight in the incubator for proper attachment. Cells were exposed to different concentrations (10–0.1 μM) of FMU200 for 24 or 48 h. At the end of the incubation period, MTT reagent was applied to each well at a final concentration of 5 mg/mL and the plate was placed in a humidified incubator at 37 °C with 5% of CO₂ for a further 3 h period. Formazan salts were dissolved in DMSO and the colorimetric determination of the reduction of MTT was determined at 570 nm wavelength using the spectrophotometer SpectraMax^® i3. Control cells treated with maintenance media were considered to be 100% viable.

To investigate the neuroprotective potential of the compound, SH-SY5Y (differentiated and undifferentiated) cells were seeded at a density of 2 × 10⁴ per well in a 96-well dish and left in the incubator overnight. Next, the medium was replaced with different concentrations (1 or 0.1 μM) of FMU200 for 30 min before adding 100 μM of 6-OHDA stabilized with 0.02% of ascorbic acid to avoid auto-oxidation of 6-OHDA. After 24 or 48 h, the treatment was removed, and MTT (5 mg/mL) was added for 3 h. Following the MTT removal, DMSO was used to dissolve the formazan salts and the OD was evaluated at 570 nm using a spectrophotometer (SpectraMax^®).