Cytotoxicity of Amyloid SMT Aggregates

To study cytotoxicity of amyloid SMT aggregates, the protein was lyophilized using a FreeZone 1l lyophilizator (Labconco). 1% trehalose was used as a stabilizing agent. SMT quality and any degradation after lyophilization were monitored using 7% SDS/PAGE [25 (link)]. Cytotoxicity was evaluated with smooth muscle cells isolated from bovine aorta as described in [29 (link)] using crystalline violet assay [30 (link)]. The cells were seeded into 96-well cell culture plates (Greiner) at density of 3000 cells per well. Cell were cultured in DMEM/F12 Nutrient Mixture (Sigma–Aldrich) with 10% FBS (Gibco), 40 μg/ml gentamicin sulfate (Sigma–Aldrich) to the confluent state, at 37°C in an atmosphere containing 5% CO₂. After the confluent formation, cells were incubated in the DMEM/F12 without serum for 2 h. SMT was added in the molecular or aggregated form. F-actin was used as a control. Cytotoxicity was estimated from the difference between absorbance in the experiment and background to the difference between the control absorbance and background in 72 h incubation. Absorbance was proportional to the number of live cells. The measurements were performed using an Infinite F200 plate reader (Tecan).