Experiments were performed to determine whether the designed peptides had hemolytic activity. RBCs from human blood were extracted (removing white blood cells and platelets), suspended in nutrient medium at a concentration of 106 RBC/μl, and stored at 4°C. Peptides were tested for hemolysis at a concentration range of 0.25 to 128 mg/liter, using a 10-fold RBC dilution (105 RBC/μl), in a solution made up to 100 μl using phosphate-buffered saline (PBS). A positive (lysis) control consisting of RBCs lysed using distilled water (DW) and a negative control consisting of RBCs incubated in PBS were also prepared. These peptide-RBC solutions and controls (12 tubes total) were incubated at 37°C for 1 hour, followed by centrifugation at 3000 rpm for 5 min. The supernatant (80 μl) was pipetted and introduced into a polystyrene 96-well plate containing 80 μl of DW (160 μl total). Using colorimetry, the absorbance difference Δabs of all wells was calculated as followsΔabs=absorbance (570nm)absorbance (620nm)

Absorbance at 570 nm is hemoglobin specific. Subtracted absorbance at 620 nm is nonspecific. The Δabs values of all wells were compared to a standard curve to determine percentage hemolysis. The steps for this hemolysis assay are depicted in fig. S13. For any given AMP, the entire protocol described in this section was repeated in triplicate to determine hemolytic activity.

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