Dimethyl sulfoxide solution

We carried out a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate cell proliferation (viability) and metabolic activity. We added HDFs (5 × 10⁴) to each well of a 24-well plate. The cells were incubated at 37 °C in fresh culture medium in an incubator with 5% CO₂, the culture medium was removed, 200 μL of 0.5 mg/mL MTT solution (Boehringer, Mannheim, Germany) was added to each well, and the mixture was incubated at 37 °C for 3 h. The substrate medium was removed, 200 μL of dimethyl sulfoxide solution (Sigma) was added to each well, and the OD at 570 nm was read using an ELISA reader (Bio-Rad, Hercules, CA, USA).

A total of 500 mg of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) powder (Sigma-Aldrich, USA) was dissolved in 100 ml of PBS to a final concentration of 5 mg/ml (0.5% MTT). The solution was stored in the dark at -20°C. A549 cells in the exponential growth phase were seeded in 96-well plates (Coring, USA) at 5×10³ cells (100 µl) per well and then incubated at 37°C in 5% CO₂ for 24 hours. Different concentrations of P3, Caerin 1.1, and Caerin 1.9 were added to the 96-well plates to reach final concentrations of 1, 5, 10, 15, 20, 25, 30, and 40 μg/ml. The experiment was done in triplicate and included control wells. The cells were cultured overnight. Once the drug’s effect was visible under the microscope, 10 µl of MTT solution was added to each well, and the cells were cultured for another 4 hours. Next, the supernatant was carefully aspirated, 150 µl of dimethyl sulfoxide solution (Sigma-Aldrich, USA) was added to each well, and the 96-well plates were placed on a shaker at low speed for 10 minutes. The absorbance at 570 nm was measured in an enzyme-linked immunosorbent assay plate reader (Thermo Scientific, USA). GraphPad Prism v9.3.0 was used calculate the half-maximal inhibitory concentration (IC₅₀) of each drug and the survival rate (%).