Hemolytic activity assay

Erythrocytes were isolated from mouse blood cells by centrifugation at 1,000×g for 10 minutes, followed by washing three times with PBS. Hemolytic activity was evaluated as the method described previously.21 Briefly, erythrocytes (final concentration 4% v/v) were treated with myristoyl-CM4 or CM4 for 1 hour at 37°C followed by centrifugation at 1,000×g for 5 minutes. The absorbance of the supernatant was measured at 414 nm, with 0.1% TritonX-100 (v/v) and PBS w as 100% and 0% hemolysis controls, Melittin was also used as hemolytic control. The percentage of hemolysis was calculated as: (A_peptide−A_PBS)/(A_TritonX-100−A_PBS)×100%, where A is the absorbance. Data were reported as the mean±standard error of the mean (SEM) of four-to-six independent experiments.