Donor horse serum

F. nucleatum PK1594, P. gingivalis ATCC 33277, S. sanguinis NC02863 and A. naeslundii 17233 were grown in Wilkins-Chagren broth (Oxoid, Basingstoke, Hampshire, UK), and incubated at 37°C for 24 h under anaerobic conditions (N₂ 85%, H₂ 5%, CO₂ 10%). S. sanguinis and A. naeslundii were transferred to Wilkins broth enriched with 2% sucrose (Sigma, Rehovot, Israel) and cultured under anaerobic conditions for an additional 24 h. F. nucleatum and P. gingivalis were transferred to Wilkins broth and incubated for an additional 24 h under anaerobic conditions. The bacteria were then centrifuged (4,000 rpm, 15 min) and suspended in gingival crevicular fluid (GCF)-simulating medium [46] (60% RPMI medium, 40% donor horse serum (Biological Industries, Beit Ha’emek, Israel)) enriched with 5 µg/mL hemin (Sigma) and 0.5 µg/mL menadione (Sigma). The bacterial suspensions of S. sanguinis, A. naeslundii and P. gingivalis were adjusted spectrophotometrically to 10⁹ cells/mL, and that of F. nucleatum was adjusted to 10⁸ cells/mL [47–50].

The H. pylori strain 26695 was obtained from the ATCC (American Type Culture Collection, 700392). The bacteria were grown on trypticase soy agar (TSA) plates, supplemented with 5% horse donor serum (Donor Horse Serum, Biological Industries, Israel), the nutrient supplement Vitox (2% Oxoid Limited, Wade Road, Basingstoke, Hampshire, UK) and the selective supplement Dent (0,2% Oxoid Limited) for 24 h under low oxygen conditions (5% CO₂, 70% humidity) at 37°C.