Hemolytic activity assay

The hemolytic activity of NK-lysin peptides was studied using bovine red blood cells (RBCs) as described previously [31]. Briefly, 10 ml of cattle blood samples were collected into syringes containing acid citrate dextrose anticoagulant and transferred to 50 ml tubes. Blood samples were centrifuged (1000 × g for 10 min) and plasma and buffy coat were removed. Resulting RBC pellets were washed twice with PBS and resuspended in PBS to obtain 2.5% hematocrit. One hundred microliter aliquots of RBC suspensions were placed into a 96-well plate and mixed gently with 20 μl of each of the diluted NK-lysin peptides (5 and 30 μM final concentration, L_Exp). Samples were incubated at 37°C in a humidified atmosphere for 60 min. The plate was centrifuged (1000 × g for 5 min) and 100 μl of each supernatant was transferred to a new 96-well plate and release of hemoglobin (from lysed RBCs) was monitored using a microplate reader by measuring the absorbance at 405 nm and 570 nm wavelengths. Triton X-100 (0.1% (v/v) final concentration, L_Tx100) -treated RBC were used as a positive control (100% lysis) and PBS-treated RBCs (L₀) were used as a negative control. The percentage of hemolysis was calculated by using the following formula (L_Exp-L₀)/L_Tx100-L₀) × 100 as described previously [31]. Means and standard error of mean were calculated from two independent experiments.