MTT reduction assay

The cytotoxicity of αS species was assessed on SH-SY5Y cells and primary rat cortical neurons seeded in 96-well plates, 24 h after their addition to the CM at various concentrations (0.03, 0.3, and 3.0 μM), by the MTT assay^37,58. SH-SY5Y cells were treated for 24 h with αS species (M, OA*, and OB*) derived from N-acetylated αS protein at 0.3 μM. In a set of experiments, LPS-free αS species (M, OB*, and LF) were compared to normal samples, by adding them to the CM of SH-SY5Y cells seeded on glass coverslips for 24 h at 0.3 μM. In a set of experiments, αS species were added to the CM of SH-SY5Y cells at 0.3 μM for different lengths of time (0, 1, 3, 5, and 24 h) and the MTT assay was assessed. In another set of experiments, OB*, SF, and LF at 0.3 μM were added to the CM of SH-SY5Y cells and, following 30 min of treatment, A11 or OC antibodies (in a molar ratio of 1:2.5) (AHB0052 Thermo Fisher Scientific and AB2286 Sigma-Aldrich, respectively), were added to the extracellular medium for 24 h and the MTT assay was assessed. A11 and OC antibodies alone were also analyzed as control. After treatment, the CM was removed, cells were washed with PBS and the MTT solution was added to the cells for 4 h. The formazan product was solubilized with cell lysis buffer (20% sodium dodecyl sulfate (SDS), 50% N, N-dimethylformamide, pH 4.7) for 1 h. The absorbance values of blue formazan were determined at 590 nm. MTT tests were achieved using Microplate Manager® Software (Biorad, CA, USA). Cell viability was expressed as the percentage of MTT reduction in treated cells as compared to those untreated.