Quantifying Cell Proliferation and Death

Cell proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT) assay as describe before20 (link). Cell death was monitored by propidium iodide (PI) staining. Briefly, cells were seeded at 3000 cells/cm² in 96-well plate and incubated at standard conditions. After 12 h, the cells were treated with scorpion peptides. Forty-eight hours after treatment, cells were incubated with MTT solution (Roth, Karlsruhe, Germany) (5 mg/ml) or PI (Sigma, Germany) (3 μg/ml), at 37 °C, 5% CO₂. After 4 h MTT incubation or 10 min of PI incubation, images were obtained using with Olympus X71 microscope (Olympus, Hamburg, Germany) with a long-distance 10 × objective. Exposure time was equal in different groups. Images were taken with cell- F software (Olympus). Cells with 4 h MTT incubation were then lysed with 100 μl acidic isopropanol. OD values were determined with a SLT spectra devise (Crailsheim, Germany) using Tecan × Fluor4 software for quantification.