Brucella broth media

The bacterial strains used in this study are listed in Table 1. H. pylori 26,695 wild-type strain (WT, ATCC 700392; CagA⁺ VacA⁺) and clinical strains were first grown on sheep blood (10% w/v) agar plates containing 10 μg/mL vancomycin (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 48 to 72 h at 37 °C under microaerophilic conditions (5% O₂, 10% CO₂ and 85% N₂). To switch the growth condition from the solid to the liquid media, bacteria were scraped off from the agar plates and resuspended in Brucella Broth media (BD Biosciences, Franklin Lakes, NJ, USA) with 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit Haemek, Israel), 1% IsoVitalex (Dr. Plate, Taipei, Taiwan). The flasks were then placed in an anaerobic chamber with shaking at 140 r.p.m and cultivated for up to 72 h under a microaerophilic condition at 37 °C.

H. pylori OMVs were isolated from bacterial culture supernatants using a method described by Horstman et al. with some modifications [62 (link)]. H. pylori 26,695 and clinical strains were inoculated with an OD₆₀₀ of 0.05 and grown in 25 mL Brucella Broth media (BD Biosciences). Various H. pylori strains were harvested after 60 h of bacterial growth. After the removal of bacterial cells by centrifugation (4000× g, 10 min, 4 °C) in a centrifuge 5810R (Eppendorf, Hamburg, Germany) with an A-4–62 rotor, supernatants were filtered through a 0.45 μm filter and then centrifuged at 200,000× g (2 h, 4 °C) in an ultracentrifuge CP80WX with a P70AT rotor (Hitachi) to collect OMVs. Pellets were suspended in 100 μL of 20 mM Tris-HCl buffer (pH 8.1–8.2) and used as the OMV preparation (stored at −20 °C). A bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin (BSA) as a standard was used to measure the protein concentration.

H. pylori 26,695 strain was inoculated with an initial optical density at 600 nm (OD₆₀₀) of 0.1 in Brucella Broth media (BD Biosciences) and incubated with a constant rotation for up to 3 days. The OD₆₀₀ values were measured at various time points.