2.5. Microbial Cell Viability Assay

Bacteria cell viability was determined while using the BacTitre-Glo^TM Microbial Cell Viability Assay (Promega, Fitchburg, WI, USA), as per the manufacturer’s protocol. Briefly, infected media was removed from HBE epithelial cells and incubated with an equivalent amount of equilibrated BacTitre-Glo^TM reagent (1:1 ratio) for five minutes in a Greinier opaque luminescence 96-well plate (Sigma-Aldrich, St. Louis, MO, USA). Luminescence was read on a SpectraMax i3X Multi-Mode Assay Microplate reader (Molecular Devices, San Jose, CA, USA). Raw luminescence units (RLU) of non-infected HBE epithelial cells were used to subtract background from infected wells. The background-subtracted luminescence values were normalized to infected HBE epithelial cells incubated with media alone (no peptide or antibiotics) to give normalized bacterial growth (%). Significance was calculated while using one-way ANOVA comparing the mean of infected HBE epithelial cells incubated with media alone to cells incubated with peptide. The incubation with water or media alone was used as a positive control for bacterial growth. Incubation with tobramycin (10 µg/mL) was used as a negative control for bacterial growth. The confirmation of infected and non-infection wells was verified via the microbial viability assay, as well as through streaking on TSB agar plates and incubation for 16 h at 37 °C.