A bacterial stimulation assay for bronchoalveolar lavage immune cells from young children with cystic fibrosis

Cystic Fibrosis (CF) is primarily a progressive lung disease, characterized by chronic pulmonary infections with opportunistic pathogens. Such typically commence early in life, producing an inflammatory response marked IL-8 chemokine production and neutrophilic infiltration, major contributory factors CF progression. Studying this inflammation, especially critical for developing new strategies preventing or slowing disruption to the structural integrity of airways. However, evaluating immune responses bronchoalveolar lavage (BAL) cells from children faces technical challenges, including contamination carried due pre-existing low cell number availability. Here, we describe technique preparing BAL young using those bacterial stimulation assay. Initial antibiotic treatment proved essential resident bacteria overgrowing cultures, non-specifically activating cells. ACTB, identified as optimal reference gene, was validated accurate analysis gene expression these Pseudomonas aeruginosa Staphylococcus aureus were used stimulants evaluate CF. Addition gentamicin prevented overgrowth, although if added after 3 hours culture extremely variable resulted, causing suppressive effect some cultures. 1 hour completely effect. This then able reproducibly measure S. P. aeruginosa, co-stimulation both bacteria.