Fresh venous blood from healthy donors was collected in tubes containing lepirudine (50 µg mL−1). Then, 50 μL of blood was transferred to a round-bottom 96-wells plate containing 150 μL of peptides previously diluted in RPMI-1640- GlutaMAX-I without phenol red (Gibco, Thermo Scientific, San Jose, CA, USA). Diluted (1:4) blood was used as a negative control. Blood (50 μL) mixed with 150 µl 5% Tween-20 was used as a positive control. After 1 h incubation at 37 °C and 5% CO2, the plate was centrifuged at 800 g, 150 μL of each sample was transferred to a flat-bottom 96-wells plate, and the absorbance at 450 nm was measured. The percentage of hemolysis was calculated following the formula (1) reported below:
To evaluate the hemolytic effect of the peptides on erythrocytes, blood was collected as reported above and centrifuged at 250 g for 10 min, plasma was discarded, and red blood cells were washed with 150 mM NaCl, 10 mM Tris, pH 7.4 for three times. The pellet was diluted 100 times in 150 mM NaCl, 10 mM Tris, pH 7.4. One-hundred microliters of this solution were added to a round-bottom 96-well plate containing 100 μL of peptides. After 1 h incubation at 37 °C and 5% CO2, the plate was centrifuged, and the absorbance at 450 nm was measured. The percentage of erythrocyte lysis was determined as above.
To evaluate the hemolytic effect of the peptides on erythrocytes, blood was collected as reported above and centrifuged at 250 g for 10 min, plasma was discarded, and red blood cells were washed with 150 mM NaCl, 10 mM Tris, pH 7.4 for three times. The pellet was diluted 100 times in 150 mM NaCl, 10 mM Tris, pH 7.4. One-hundred microliters of this solution were added to a round-bottom 96-well plate containing 100 μL of peptides. After 1 h incubation at 37 °C and 5% CO2, the plate was centrifuged, and the absorbance at 450 nm was measured. The percentage of erythrocyte lysis was determined as above.