Campygen gas mix

Human MKN-28 cells (JCRB, #0253) were originally isolated from gastric adenocarcinoma. The Caco-2 cells (ATCC HTB-37) were obtained from a human colon adenocarcinoma. Both cell lines have been extensively used over the last twenty years as models for studying the gastrointestinal barrier. Cells were cultured in 6-well plates with RPMI1640 or DMEM medium, respectively, containing 4 mM glutamine (Invitrogen, Karlsruhe/Germany), and 10% FCS (Invitrogen, Karlsruhe/Germany) [68 (link)]. H. pylori strains 26695, P12 and their mutants were grown on horse serum GC agar plates supplemented with nystatin (1 μg/mL), vancomycin (10 μg/mL) and trimethoprim (5 μg/mL), and if necessary with 4 μg/chloramphenicol per mL. Growth was performed for 2 days at 37 °C in anaerobic chambers containing a CampyGen gas mix (Oxoid, Wesel/Germany) at 37 °C [69 (link)]. H. pylori was harvested and resuspended in phosphate buffered saline (PBS, pH 7.4) using sterile cotton swabs (Carl Roth, Karlsruhe/Germany). The bacterial concentration was measured in a spectrophotometer as optical density (OD) at 600 nm (Eppendorf, Hamburg/Germany). Infections were carried out at a multiplicity of infection (MOI) of 50 [70 (link)]. All infection assays were done in triplicates.

Campylobacter jejuni strains 81-176 and NCTC11168 were grown on Campylobacter blood-free selective Agar Base (Oxoid, Wesel, Germany) plates containing Campylobacter supplement (Oxoid) for 2 days at 37 °C in an anaerobic jar together with a CampyGen gas mix (Oxoid). For CFU counting the bacteria were plated on Mueller–Hinton (MH) agar (Oxoid) with 10 µg/mL vancomycin. E. coli BL21 (NEB, Ipswitch, USA) was cultivated in Terrific Broth (TB) medium containing 20 g/L tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 0.072 M K₂HPO₄ and 0.017 M KH₂PO₄ at 37 °C under agitation.