Amyloid Fibrils Cytotoxicity Assay

The toxicity effects of all the amyloid fibrils were tested on the SH-SY5Y human neuroblastoma cell line using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) colorimetric assay. The cells were grown in 1:1 mix of Dulbecco's Modified Eagle Medium (DMEM) and Ham's F-12 growth media with 5% Fetal Bovine Serum (FBS) (Gibco) containing L-Glutamine and Phenol Red in 75 mm³ T-75 flasks at 37 °C in 5% carbon dioxide (CO₂) environment till they achieved 70–90% confluency. After 24 h, approximately 2,500 cells were transferred into each reaction well of 96 well transparent tissue culture plate (Corning^® 96 Well TC-Treated Microplates). Once the cells were stabilized, 20 µl of samples were added in each well containing 100 µl culture media. Before the fibrils were added to the cells, they underwent a washing protocol to remove excess/unbound drug compounds as described previously (Palhano et al., 2013 (link)). The samples were added into the cell assay and incubated for 20 h to test their toxic effects. After incubation, 10 µl of the stock MTT reagent (5 mg/ml) was added to each well. After 4 h of conversion into the formazan product, DMSO was added to dissolve the purple crystals left in the dark for 1 h before the measurement of absorbance at 570 nm with 630 nm as a subtracted reference wavelength.