Preparation of Helicobacter pylori culture filtrate

Helicobacter pylori culture filtrate (Hpcf) was prepared as described by Cuomo et al. [36 (link)]. In detail, culture filtrate of H. pylori P12 strain was prepared by culturing the bacterium on selective Columbia agar (Oxoid, Basingstoke, Hampshire, UK) supplemented with 7% (v/v) of defibrinated horse serum (Oxoid, Basingstoke, Hampshire, UK) and antibiotic mix (DENT; Oxoid). Bacteria plates were incubated for 3–4 days at 37°C in a 10% CO₂ atmosphere. After growth, bacteria were scratched using brain heart infusion (BHI; Oxoid, Basingstoke, Hampshire, UK) and measured. 2 x 10⁷ bacteria were cultured in DMEM (Microtech, Naples, Italy) supplemented with 10% FBS (Microtech, Naples, Italy) and incubated at 37°C in a 10% CO₂ atmosphere for 24 hours. Finally, bacterial suspension was centrifuged at 10,000 g for 10 minutes and the supernatant was filtered by using a 0.22 μm filter.