Activity metrics for peptides in complete set

Spreadsheet containing 50% Inhibitory concentration (IC50) and microbiocidal concentration (MBC) of the complete set of peptides against <i>S. aureus, P. aeruginosa </i>and <i>C. albicans. </i><br>50% hemolytic concentration (EC50) is also included. For each peptide mean and standard error are provided over four independent experiments. <i><br></i>The new metric ODvis which is used to determine solubility of the peptides is also included.<b>Antimicrobial</b><i> </i><b>assays (IC₅₀ and MBC)</b><i></i>Planktonic cultures were obtained by inoculating single colonies from 18-20 hour old agar cultures into 10 ml of sterile broth, followed by 20-hour incubation. Test organisms were isolated by centrifugation of 1ml of the culture at 2000<i>g</i> for 3 minutes (MiniSpin^®; Eppendorf, Germany), and resuspending the pellet in 1 ml of fresh sterile broth, before further dilution in sterile broth, until an optical density at 600nm (OD₆₀₀) of 0.8 was obtained. This was diluted 1:50 in sterile broth, resulting in an inoculum of approximately 10⁶ colony-forming units per ml (CFU/ml). Peptide stock solutions of 800 µM were prepared by dissolving solid peptides (provided in pre-weighed aliquots by the supplier) in phosphate buffer solution (PBS) containing 5% (v/v) dimethyl sufoxide (DMSO). Further dilutions of the stock peptides were prepared (at double the final test concentrations of 0.6-400 µM) in PBS in 5% (v/v) DMSO. All peptide stocks and dilutions were prepared in polypropylene centrifuge tubes. 100 µl of the test inoculum was transferred into the wells of a polystyrene 96 well microtiter plate and 100 µl of each of the peptide concentrations were added into each test well. Each polystyrene plate was then aerobically incubated at 37 °C for 18-20 hours without shaking, before measuring OD₆₀₀ using a spectrophotometer plate reader (Nanostar; BMG Labtech, Ayelsbury). A sigmoidal curve was fitted to the resulting data, from which the concentration resulting in a 50% inhibition of bacterial growth (IC₅₀) was obtained. The mean IC₅₀ for each peptide was calculated from the results of four independent experiments, with a single technical replicate each. Minimum biocidal activity (MBC) for each peptide was assessed by removing 5µl samples from each well following the IC₅₀ determination, then adding these samples to agar and incubating them at 37°C for 18-20 hours. MBC was considered to be the lowest concentration of each peptide in which no microorganism growth was observed across three technical replicates. The mean minimum biocidal concentration (MBC) was calculated using data from four independent experiments. <b>Determination of hemolytic activity (EC50)</b> Erythrocytes from defibrinated horse blood were prepared by centrifugation at 2000 <i>g</i>, 4 °C for 10 minutes. The pellet was resuspended in PBS and the cells washed a further two times by centrifugation (2000 <i>g</i>, 4 °C for 10 minutes) and resuspension PBS a further 2 times. An erythrocyte test suspension was prepared by resuspending cells in PBS (20% v/v) and was stored at 4 °C, prior to use. For assays, a 1% v/v cell suspension in PBS was prepared and 100 µl aliquots transferred to polypropylene v-bottomed 96 well microtiter plates. Dilutions of the stock solution of each peptide were plated into a separate polypropylene 96 well microtiter plate (at double the final test concentrations), and 100µl of each peptide solution was then transferred into 96 well plate containing the test cell suspensions, before incubation at 37 °C for 1 hour. After incubation, non-lysed cells were removed by centrifugation at 2000 <i>g</i>, 4 °C for 5 minutes. Finally, 100µl of the supernatant was transferred to a flat bottomed 96 well plate, and the hemoglobin content of the supernatant was assessed by measured OD₄₅₀using a spectrophotometer plate reader (Nanostar; BMG Labtech, Aylsbury, UK). Sigmoidal curves were fitted to the resulting data, from which the concentration of peptide resulting in 50% lysis of erythrocytes was determined. Mean EC50 was calculated using data from four independent experiments, each composed of a single technical replicate. <b>Stock solution turbidity (OD_vis)</b>Peptide stock solutions were prepared at 800 µM in PBS with 5% v/v DMSO. 100µl of each peptide solution was then transferred into 96 well plate and OD_vis was then assessed by measuring OD₆₀₀ using a spectrophotometer plate reader (Nanostar; BMG Labtech, Ayelsbury). Mean OD_vis was calculated using data from four repeat experiments, each composed of a single technical replicate.