Melittin

Human red blood cells (hemoglobin concentration (Hb) 150–160 g L⁻¹) were collected from apparently healthy patients in EDTA tubes. 600 μL of EDTA-blood were washed in TBS buffer (10 mM TRIS, 150 mM NaCl) and centrifuged at 1500g for 1 min until the supernatant became colourless/colourless. The final pellet was diluted to 5 mL with TBS buffer. Hundred microliters of this cell suspension were pipetted into sterile Eppendorf tubes together with twofold serial dilutions of each compound to a final volume of 200 μl. Final concentrations ranged between 2500–9.75 μg mL⁻¹. Following incubation for one hour at 37 °C, samples were centrifuged at 1500g for 1 min to precipitate the red blood cells. All supernatants were transferred to sterile 96-well plates for the measurement of their direct optical density (OD) at 565 nm wavelength (Multiskan FC microplate reader, Thermo Scientific). Melittin (Bachem) at concentration of 50 μg mL⁻¹ and TBS were used as positive (100% hemolysis) and negative (no hemolysis) controls, respectively. Haemolytic effect of each peptide at each concentration was calculated as follows: hemolysis effect = (compound OD_565nm − TBS OD_565nm) × 100/(Melitin OD_565nm − TBS OD_565nm).