Peptide Cytotoxicity and Hemolysis Assays

To assess cytotoxicity of optimized peptides in mammalian cells, an immortalized human keratinocyte cell line (HaCaT) was used. Cells were grown to 80% confluency in 24-well culture dishes and washed with PBS (Thermo Fisher). Each peptide was diluted in fresh Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), to desired concentrations corresponding to MIC experiments and incubated with cells for 16 h in triplicate at 37°C, 5% CO₂. After incubation, the cells were washed with sterile PBS, followed by a covered incubation with 4 μM ethidium homodimer (Molecular Probes) for 30 min at room temperature. Fluorescence was measured using the Synergy H1 Microplate Reader set to 528 nm excitation, 617 nm emission, and a cutoff value of 590 nm. Saponin (0.1% wt/vol, Sigma) was added to each well followed by a covered incubation for 20 min at room temperature, followed by another measurement on the Synergy H1 Microplate Reader using the same settings to normalize readings to the number of cells per well and determine the percent cytotoxicity. Peptides were characterized as cytotoxic at a particular concentration if incubation with that concentration of peptide resulted in more than 20% cell death after 16 h, which corresponded to the percent cytotoxicity in HaCaTs treated with the vehicle control under the same conditions.

To determine if peptide variants were hemolytic, fresh defibrinated whole sheep blood (Hardy Diagnostics) was washed three times in cold PBS, and washed blood was diluted 1:25 in PBS. Treatments were applied at a 1:10 ratio of sample:blood, where the sample was either 10% Triton, PBS, or a specific peptide concentration corresponding to concentrations used in the MIC assays. Treatments were incubated with the blood for 1 h at 37°C. Following incubation, samples were centrifuged at 0.4 × g for 10 min at 4°C, and the absorbance of the supernatants at 450 nm was measured on the Synergy H1 Microplate Reader to measure the release of hemoglobin. Percent hemolysis was quantified by normalizing to the negative and positive controls.