2.9. Co-Culture Conditions of Human Gastric Cancer Cell Lines and H. pylori Strains In Vitro

The human gastric adenocarcinoma cell lines AGS (ATCC #CRL-1739) and MKN45G [40] were cultured in RPMI 1640 GlutaMAX (GIBCO, Life Technologies, Carlsbad, CA, USA) without antibiotics, but supplemented with 6% heat-inactivated fetal calf serum (GIBCO) at 37 °C and 5% CO₂. Confluent cultures were trypsinized, and 5 × 10⁶ cells were added to 10-cm tissue culture dishes and allowed to reconstitute overnight, reaching approximately 80% confluence.

Suspensions of freshly harvested H. pylori strains SS1 or J99 were adjusted to an optical density of 600 nm = 0.25 in RPMI 1640 GlutaMAX without fetal calf serum. Then, 10 mL of bacterial suspensions (corresponding to 1.0 × 10⁹ CFU) or the corresponding buffer controls were added to tissue culture dishes containing adherent MKN45G or AGS cells. After 6 h of incubation at 37 °C, the co-cultures were washed with PBS and cells were harvested using a cell scraper and collected three times in 300 µL 0.1 M Tris pH 8.1, 10 µg/mL trasylol, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The cell pellets were collected by centrifugation at 12.000× g for 30 min at 4 °C and immediately lysed by re-suspension in 900 µL of lysis buffer (0.1 M Tris pH 8.1, 0.5% (w/v) deoxycholat, 1% (v/v) NP40, 0.1% (w/v) SDS, 10 mM EDTA, 1 mM PMSF, and 10 µg/mL trasylol). Lysis proceeded on ice for 60 min with short pulses of vortexing every 20 min. Supernatants were isolated by centrifugation at 15.000× g for 20 min at 4 °C.

Detection of uPAR expression in these detergent lysates was assessed by SDS-PAGE of 10 µL of lysate (~6 × 10⁴ cells) followed by electroblotting onto PVDF membranes. Excess protein binding sites were blocked by incubation with 2% (w/v) skim milk powder. Visualization of uPAR expression was subsequently accomplished by first an overnight incubation at 4 °C with 0.1 µg/mL of the primary rabbit anti-uPAR pAb and a second 1-h incubation at room temperature with a 5000-fold dilution of secondary HRP-conjugated swine anti rabbit-Ig pAb (Code P 0217, DAKO, Glostrup, Denmark). Specificity was validated by pre-incubation of the primary rabbit anti-uPAR antibody with a 10-fold molar excess of purified soluble uPAR expressed in Drosophila S2 cells [41], before addition to the PVDF membrane. Equal loading conditions were assessed after stripping the membrane and reprobing the blots with a mouse anti β-actin mAb (code: mAbcam 8226; Abcam, Cambridge, UK).