Single representative colonies were inoculated into fresh LB broth and incubated overnight at 37°C. Media were supplemented with relevant antibiotics (Sigma) at concentrations: kanamycin (50 μg/ml), tetracycline (20 μg/ml), ampicillin (50 μg/ml) and chloramphenicol (20 μg/ml). Motility measurement Motility was Linsitinib concentration assayed in Heart Infusion broth with 0.25 % agar (Difco) and on Swarm agar (Statens Serum

Institute, DK) as described [43]. Expression of flagella antigens Serotyping was performed as previously described [43]. Western blot was performed using NuPAGE™ 12,5% Tris–HCl gels (Novex) as instructed by the manufacturer and specific flagella antisera (H:i, H:2 or H:p,g), (Statens Serum XMU-MP-1 ic50 Institute (SSI), Denmark). Demonstration of flagella by electron microscopy To demonstrate flagella, bacteria were negatively stained with uranyl acetate 2% and examined by transmission electron microscopy at an instrumental magnification of 27500. Adhesion and survival properties in vitro Comparison of in vitro adhesion, invasion (uptake) and survival of bacteria inside cells was performed using the epithelial cell line Int407 and the macrophage-like cell line, J774A.1, as previously described [46]. Before experiments with macrophages, bacteria were opsonised with 10 % heat treated foetal calf serum (Invitrogen) for

30 min at 37°C prior to addition to the cells. Infections were performed at a multiplicity of infection (m.o.i.) of 10:1 with the macrophage cell line and 100:1 with Int407 cells. For all experiments, C59 wnt cell line cells were centrifuged at 1500 rpm for 2 min. immediately after infection to allow close contact GBA3 of the bacteria with the cells. Each bacterial strain was assayed in triplicate and experiments were

repeated once. Cytotoxicity Cytotoxicity to macrophages was determined by release of lactate dehydrogenase (LDH) by the monolayers into supernatants using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega G1780). Results were expressed as the percentage of LDH released by infected monolayers compared to LDH release by lysis buffer treated (lysed) monolayers at 24 hours [(A495 test sample – A495 medium control) / (A495 macrophage+lysis buffer – A495 medium+lysis buffer)] × 100. Induction of oxidative radicals (chemiluminescence) The method described by Chadfield and Olsen [47] was used. Opsonized zymosan (Sigma) and phorbol myristate acetate (PMA)(Sigma) was used as positive control stimuli. A Lucigenin probe (Sigma) dissolved in DMSO (Sigma) and diluted in Hanks balanced salt solution (HBBS) (Gibco Life Technologies) to final assay concentrations of 150 μg/ml was used. Cells used in the assays were J774A.1. The luminometer (AUTOLUMAT LB 953, Berthold) was set at 37°C. The reading intervals were minutes and the duration of the assays were 300 minutes.