Trypticasetm soy agar with 5 sheep blood tsaii

The human gastric cancer cell lines Hs746T (C0023003; AddexBio, San Diego, CA, USA), MKN74 (a kind gift from Carla Oliveira, University of Porto) and NCI-N87 (ATCC^® CRL-5822^TM; ATCC, Manassas, VA, USA), referred as HsEUR, HsEAS and HsAFR, respectively, were cultured in DMEM with Glutamax (Gibco, Dublin, Ireland) supplemented with 10% fetal bovine serum (LabClinics, Barcelona, Spain) and 100 U-100 µg/mL penicillin-streptomycin sulfate (Gibco, Dublin, Ireland), at 37 °C, under a 5% CO₂ humidified atmosphere.
H. pylori strain 26695 (ATCC 700392) was obtained from ATCC while H. pylori strain J99 (ATCC 700824) was a kind gift from Celso Reis, University of Porto. These strains will be further referred as HpEUR and HpAFR, respectively. They were cultured in Trypticase^TM Soy Agar with 5% Sheep Blood (TSAII; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at 37 °C under microaerophilic conditions (GENbox microaer; bioMérieux S.A., Marcy-l’Étoile, France) for 48 h.

H. pylori strain 26695 (ATCC 700392, cagPAI+, vacA s1/m1) was obtained from ATCC (LGC Standards, United Kingdom), and H. pylori strain 60190 (ATCC 49503 cagPAI+, vacA s1/m1) and its respective 60190CagA^-, 60190CagE^-, and 60190VacA^- mutants were a kind gift from Professor John Atherton (University of Nottingham). Strains were cultured in Trypticase^TM Soy Agar with 5% Sheep Blood (TSAII; Becton, Dickinson and Company) at 37°C under microaerophilic atmosphere (GENbox microaer; bioMérieux S.A.) for 48 h, as previously described (Costa et al., 2016 (link)). For the production of conditioned media, bacteria were grown in F12 medium (Gibco) supplemented with 1x cholesterol (Gibco) under microaerophilic conditions at 37°C with constant rotation (150 rpm) over 24 h. Subsequently, bacterial suspensions were centrifuged at 15,000 × g, for 15 min, and the supernatants were filtered through a 0.2-μm sterile filter, and concentrated by ultrafiltration using a 10 kDa pore size Amicon (EMD Millipore). The resulting concentrated bacterial conditioned media was used as media for gastric cell cultures in comparison with F12 medium plus 1x cholesterol alone without bacteria, as control.