Tracheal aspirates were collected from 11 patients with BPD and 10 term-born control infants as previously described (21). Aspirate fluid was subjected to centrifugation at 10,000g for 1 hour to pellet cells and smaller particles. Subsequently, the supernatant fluid was subjected to ultracentrifugation (150,000g for 2 hours) for exosome extraction. Exosomes collected from individual patients within each group (BPD and control) were pooled, quantified, and resuspended in PBS prior to use in experiments. Elastase content of exosome samples were measured in duplicate using the EnzChek elastase assay kit (Molecular Probes) per the manufacturer’s suggested methods. Known concentrations of HNE were run in parallel to generate a standard curve. Exosome elastase activity was expressed in HNE equivalents.

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