Ivacaftor

Polymyxin B (Catalogue number 81334, ≥6,500 IU/mg) was purchased from Sigma-Aldrich (Australia). Ivacaftor, lumacaftor and VX-661 were purchased from (SelleckChem, USA). All other reagents were purchased from Sigma-Aldrich (Australia) and are of the highest commercial grade available.

Human or mouse neutrophils (5×10⁵) were infected with Burkholderia cenocepacia (Bc), Pseudomonas aeruginosa (Pa01), Staphylococcus aureus (Sa) or Nontypeable Haemophilus influenzae (NTHI) using a multiplicity of infection of 10 (MOI=10) in RPMI medium with 2% FBS for 45 minutes. Cells were centrifuged at 1200 rpm for 7 minutes, the supernatant was removed, and the pellet was resuspended and seeded in 24 well plates, then some neutrophils were treated with 3×10⁻⁶ M of EGTA (Sigma Aldrich, St. Louis, MO) or 10⁻⁵ M of 2-APB (Sigma Aldrich, St. Louis, USA) and incubated for 3 hours at 37 °C. Some samples were pretreated with 10⁻⁵ M DPI for 10 minutes or with or with 5×10⁻⁶ M of VX-770 (Ivacaftor, Selleckchem.com) for 60 minutes at 37 °C and maintained with the potentiator during the course of infection. To quantify intracellular bacteria, neutrophils were lysed with 0.1% Triton X-100 (Sigma Aldrich, St. Louis, MO), and free bacteria were quantified by serial dilution on LB and chocolate agar plates. Relative percent of antimicrobial killing was calculated by dividing the inverse CFU of CF with non-CF neutrophils and multiplied by 100.