Haemolysis assay

The haemolytic activity of the peptides was evaluated by determining haemoglobin release from a blood erythrocyte suspension²⁶. Briefly, erythrocytes were washed three times with 10 mM phosphate-buffered saline (PBS, pH 7.3) and were centrifuged at 1,300 × g and 4 °C for 10 min. Next, 100 μL of erythrocyte suspension diluted with 10 mM PBS (final concentration, approximately 2%) was mixed with 100 μL of each serial two-fold dilution of AMPs (final concentration, 0.5–128 μM). The mixtures were incubated at 37 °C for 1 h. After incubation, the mixtures were centrifuged at 1,300 × g for 5 min. The resulting supernatants were transferred into 96-well microtiter plates, and optical density was measured at 540 nm using a Multiskan Spectrum (Thermo Fisher Scientific, USA). Values for 0% and 100% lysis were determined by incubating the erythrocytes with 10 mM PBS and 0.1% (v/v) Triton X-100, respectively.