PI-uptake assay-fluorescence microscopy

Cells (1-5×10⁴) were seeded on Ibidi μ-Slide 8 wells and grown in 300 μl respective media for 2-3 days to a confluent layer. Propidium iodide (PI, 2 μl of 50 μg/ml in PBS, Biosource, Camarillo, CA, USA) was added to the well and cell status was checked after 5 minutes of incubation in the dark at room temperature. Then, peptide was added to the desired concentration and peptide effect was followed immediately. Pictures were taken every 5 or 15 minutes for up to 8 hours from the same section of cells. Excitation and emission wavelength were as follows: PI excitation, 535 nm and emission, 617 nm.

MTS viability assay-spectroscopy—Cell proliferation was measured by using CellTiter 96® Aqueous One Solution Cell Proliferation assay (Promega). Cells were plated in 96-well plates and grown until confluence in respective medium containing 2% serum at maximum to avoid interference with the MTS compound. Peptides were added to a final concentration of 5-100 μM. After incubation for 24 h at 37°C (5% CO₂) MTS [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-phenozine methosulfate solution (20 μl/well) was added and cells were again incubated for 2 hours at 37°C (5% CO₂). The MTS compound is bio-reduced by cells into a colored formazan product that is soluble in tissue culture medium. The quantity of the formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture. Data are calculated as a percentage of the control (untreated) samples and represent the average of three wells in one experiment which was repeated three times per cell line.