Bovine dnase 1

To quantify BrdU-labeled doublecortin (DCX)-positive neuroblasts, BrdU was diluted in sterile 1× PBS and injected intraperitoneally at 100 mg/kg in 24 hr intervals 2 days before sacrifice. To isolate neural progenitor cells for flow cytometry, tissue samples were triturated in HBSS (Invitrogen), followed by enzymatic dissociation in 0.25% trypsin (Gibco) with bovine DNase-I (Sigma-Aldrich) in a 37°C water bath for 10 min. Samples were then filtered through a 70 µm cell strainer and centrifuged for 20 min in 30% Percoll solution (Sigma-Aldrich) diluted in 1× PBS. The centrifugation separated out myelin and other cellular debris, which were subsequently removed with the Percoll solution. Pelleted cells were separated into sample tubes and pooled together for compensation controls. Each sample was incubated in Fc receptor block, fixed, and stained for surface antigens, including CD45. Next, cells were permeabilized and stained for intracellular antigens (DCX, BrdU). BrdU staining was done using the FITC BrdU flow kit (BD Biosciences). Finally, fluorescently-conjugated secondary antibodies and DAPI were added to samples before quantification on the LSR II Flow Cytometer (BD Biosciences).

Sputum samples from CF patients were kindly collected by Dr. M. Maschio at the Institute for Maternal and Child Health IRCCS “Burlo Garofolo”, Trieste, Italy. They were pooled and divided in aliquots that were stored at −20 °C. Bacterial killing assay was performed against P. aeruginosa strain RP73 in modified SCFM (Synthetic Cystic Fibrosis Sputum Medium) [18 (link),21 (link)] or in CF sputum diluted to 25% v/v in modified SCFM. D-BMAP18 or tobramycin were incubated for 4h using a water bath at 37 °C with 300 mM NaCl (to a final tested concentration of NaCl = 450 mM) and/or with a final tested concentration of 128 μg/mL of bovine DNase I (Sigma-Aldrich, St. Louis, MO, USA) in a final volume of 100 μL of SCFM or 25% CF sputum containing 106 CFU/mL. Samples were then diluted 10-fold in SCFM and 25 μL of each dilution was plated on MHA incubated o/n at 37 °C for viable colony count. Dilutions of the CF sputum alone were also plated on MHA to evaluate its contamination by the endogenous microbiota. No growth of endogenous bacteria was observed in the sputum within the indicated time.

Inhibition of biofilm formation was evaluated against the P. aeruginosa PAO1, PA03, PA05, PA07, PA08, PA09, PA10, PA14, PA21, PA22, PA31, PA35, and RP73 strains using the MTT assay as previously described by Mardirossian and colleagues [17 (link)]. Biofilm eradication assays were performed against P. aeruginosa strains PAO1, PA08, PA09, and RP73, as previously reported by Bonaventura et al. [18 (link)]. After an 18 h incubation in MH broth in 96-well flat-bottom microtiter plates (Sarstedt, Milan, Italy), the bacterial biofilms were incubated 24 h at 37 °C with different concentrations of D-BMAP18 in 50 μL of MH broth. Before peptide addition, some samples were pre-incubated for 1h at 37 °C in the absence or presence of 128 μg/mL of bovine DNase I (Sigma-Aldrich), or 10 mg/mL of N-Acetyl-L-cysteine (Sigma-Aldrich) or 1 mg/mL of Alginate lyase (Sigma-Aldrich). After incubation, the MTT assay was performed as reported [17 (link)].