, 2008). Experiments performed with viable bacteria yielded the equivalent of 5 × 108S. aureus Cowan I from a suspension with an optical density (OD600 nm) of 1.0. Staphylococci were adjusted to an estimated concentration of 2 × 108 CFU mL−1 cell culture medium and kept at +4 °C until use.

Three FACS experiments were performed as previously described, on different days in duplicates, Epacadostat and up to 5000 invasion events were counted, unless described elsewhere. Staphylococcus aureus Cowan I and S. carnosus TM 300 were measured in the same experiment as a positive control and a negative control, respectively. The arbitrary value of FITC-stained bacteria, used as a surrogate for invasion of cells, was normalized to the positive control S. aureus Cowan I to display the relative invasiveness of the tested strains to the strongly invasive S. aureus Cowan I. Purified fibrinogen (plasminogen, von Willebrand-factor and fibronectin depleted; Enzyme Research Laboratories, Ceritinib nmr South Bend, IL) was coated to a 96-well microtiter as previously described (Szabados et al., 2011). For the fibronectin binding, a precoated microtiter plate was used (BD Biocoat™ Cellware Human Fibronectin; BD, Bedford, MA). The binding experiments were performed as previously described (Szabados et al., 2011). An OD550 nm

value of 0–0.06 was interpreted as negative, 0.07–0.15 as intermediately positive (+), 0.15–0.3 as positive (++), and > 0.3 as strongly positive (+++). Staphylococcus aureus Cowan I was used as positive control for fibrinogen and fibronectin binding. A sample without bacteria 2-hydroxyphytanoyl-CoA lyase and the S. carnosus TM 300 were used as negative controls. Bacteria (1 × 108) were washed with PBS and suspended in an estimated 1 μg mL−1 FITC and incubated for 30 min. Bacteria were washed three times with ice-cold PBS. Sulfo-NHS-LC-biotin (Pierce Biotechnology, Rockford, IL) was solved at a final concentration of 0.3 mg mL−1

in PBS as previously described (Agerer et al., 2004). Samples were washed three times with PBS, mounted with embedding medium ProLong® Gold (Invitrogen) in glass slides and sealed with nail polish. The glass slides were examined using confocal microscope Leica DM IRE2 (Leica, Solms, Germany). A suspension of human urinary bladder carcinoma cells 5637 from the FACS assay was used. The lysis step was omitted and cells were centrifuged gently (1000 g) for 60 s and transferred into 500 μL D-PBS (PAA) and fixed with 500 μL glutaraldehyde 2.5% as previously described. Only three of eight strains (Stlu 12, Stlu 50, and Stlu 108) showed binding to solid-phase fibrinogen (Fig. 1a)- as seen in previous results (Szabados et al., 2011). Four of eight strains (Stlu 30, Stlu 33, Stlu 36 and Stlu 108) showed binding to solid-phase fibronectin (Fig. 1b). One strain (Stlu 108) showed binding to immobilized fibrinogen and also to immobilized fibronectin.