*5.3. Cell Viability Assay*

The cytotoxicity assays were carried out by treating the cells with different concentrations of venom in the range from 0.1 μM to 20 μM. The cytotoxicity tests were performed using the colorimetric method of cell viability analysis using the WST-1 cell proliferation reagent kit (Roche, Mannheim, Germany) according to manufacturer's instructions. Briefly, 104 cells were plated in each well of a flat bottom 96-well microtiter plate. Cells were cultured at 37 ◦C in 5% CO2 and on the day prior to analysis (70–80% confluency), culture medium from each well was exchanged to 100 μL of medium in the absence or presence of venom in different concentrations. After 24 h, 10 μL of the WST-1 reagent previously dissolved into an Electro Coupling Solution (ECS) were added to each well and incubated

for 4 h at 37 ◦C in 5% CO2. The absorbance at 450 nm of each well was measured using a microplate reader FlexStation 3 spectrophotometer (Molecular Devices, San Jose, CA, USA). The determination of the absorbance and the quantification of viable cells were calculated using SoftMax Pro (version 5.1, Molecular Devices) and Microsoft Excel software.
