4.4. Cell Viability Assay

Cytotoxicity in N2a and MC65 cells cultured and treated as described in Section 4.2 was determined using counts of viable cells 72 h after treatment based on the MTT assay (tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), which was described previously by Maezawa et al., 2007. Both N2a and MC65 cells were plated at a density of 2.5 × 10⁵ cells/well in a 96-well plate in 200 μL in the same medium described in Section 4.2. The Aβ solution was prepared in phenol red-free (OPTIMEM) and fetal bovine serum-free media as described in Section 4.3, then added to the N2a cell cultures at final concentrations of 1 μM for Aβ and 2 μM for SLF, SLF^dm, and MitoTEMPO. The MC65 cells were cultured in the same conditions in the presence and absence of TC at 1 μg/mL. The compounds were added to the cultures at 2 μM for SLF, SLF^dm, and MitoTEMPO for 9 wells and 3 rows. After 24 h of incubation, 150 μL media was removed and 5.5 μL of 2.5 mg/mL MTT (thyazolyl blue compound) was added to each well to a final concentration of 0.25 mg/mL. After 2 h incubation at 37 °C, 100 μL of solubilization buffer (0.1 N HCL in isopropanol) was added to each well and the plates were placed on a shaker at room temperature until blue crystals are dissolved. The plate was read on the plate reader at 560 nm for MTT and 630 nm for baseline subtraction. Cell viability was expressed as the percentage of viable cells.