To show that live bacterial challenge causes the release of EVs, mice were infected with Haemophilus influenzae (H. influenzae). Briefly, Hib Eagan strain was a kind gift from P. Langford (Faculty of Medicine, St Mary's Hospital, Imperial College London). Bacteria were cultured at 37°C in 5% CO2 in Brain heart Infusion broth (OXOID) supplemented with 10 µg/ml of both Hemin (10ug/ml) and Nictinamide adenine dinucleotide (NAD) (Sigma-Aldrich, UK) or on BHI agar (OXOID) supplemented with 4% Levinthals when agar was <50°C. Levinthals was made by adding 50% horse blood (TCS Biosciences) to BHI broth and heating to 70°C for 45 minutes. On cooling to 50°C, 0.7 mg/ml NAD was added and the supernatant stored at –80°C. Bacteria were cultured to an OD600 of 0.3 (approximately 1×109 CFU/ml) and stored at −80°C in 10% glycerol as single use aliquots.
Groups of mice (n = 4–5 per group) were infected i.n. with 1×107 colony forming units of H. influenzae serotype b (strain Eagan) in sterile phosphate buffered saline (PBS). Terminal anaesthesia was induced at 6, 24 and 72 hours after challenge. Mice were culled and BALF samples were collected at 6, 24 and 48 hours. Presence of EVs was assessed as detailed above.
Groups of mice (n = 4–5 per group) were infected i.n. with 1×107 colony forming units of H. influenzae serotype b (strain Eagan) in sterile phosphate buffered saline (PBS). Terminal anaesthesia was induced at 6, 24 and 72 hours after challenge. Mice were culled and BALF samples were collected at 6, 24 and 48 hours. Presence of EVs was assessed as detailed above.