2.12. Hemolytic and Cytotoxicity Assays

Hemolytic activity of Lc-def was studied using 96-well microplates. Fresh human red blood cells (hRBC) were washed three times with ice-cold PBS, pH 7.2. Serial two-fold dilutions of Lc-def and membrane-active peptide melittin from the venom of honeybees as positive control (final concentrations from 0.1 to 100 μM) were then added to hRBC in PBS (final concentration of 4% (v/v)). The suspension was incubated for 1.5 h at 37 °C. After, intact hRBC sedimentation aliquots of the supernatants containing released hemoglobin were transferred to 96-well microplates, and the absorbance at 405 nm was measured. hRBC in PBS or 0.1% Triton X-100 were employed as negative (control0) and positive (control100) controls, respectively. The percentage of hemolysis was calculated as: hemolysis(%) = ((A_sample − A_control0)/(A_control100 − A_control0)) × 100%. Experiment was carried out twice in triplicate using the same blood.

The cytotoxic properties of Lc-def and melittin as a control were investigated also by resazurin-based cell cytotoxicity assay. PBMCs and THP-1 cells were seeded in 96-well plates at 2 × 10⁶ and 1 × 10⁶ cells per well, respectively, in RPMI-1640 supplemented with 10% FBS, and kept in CO₂-incubator. After 24 h serial two-fold dilutions of compounds in culture medium were then added to final concentrations from 0.2 to 50 μM. After 24 h of incubation with the peptides, resazurin (Sigma) was added at a final concentration of 0.7 mM, and the plates were incubated overnight (16 h). Fluorescent resorufin was registered using 535/595 filter at PlateReader AF2200 (Eppendorf, Hamburg, Germany). Untreated corresponding cells were used as negative controls. The cell viability was calculated as: cell viability (%) = (F_sample/F_control) × 100%. The experiment was carried out twice in triplicate.