, 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, Selleckchem Dasatinib 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, Crizotinib manufacturer 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 Protein kinase N1 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.