Plasma plasminogen

The binding of bacteria to plasma and/or ECM proteins was analyzed as described elsewhere (Agarwal et al., 2013 (link)) with modifications. Briefly, black polystyrene 96-well microtiter plates were treated (18 h at 4°C) with each human protein [plasma fibronectin, plasma fibrinogen, plasma plasminogen, or type I collagen from human fibroblast (Sigma-Aldrich, United States); 5 μg/ml in PBS pH 7.2]. Afterward, the plates were washed (three times with PBST) and incubated (2 h at room temperature) with blocking solution (50 mM Tris–HCl – pH 8, 150 mM NaCl, 0.1% Tween 20, 3% fish gelatin). Next, 100 μl of suspensions of FITC-labeled bacteria in carbonate buffer (0.15 M NaCl, 0.1 M Na₂CO₃; pH 9.6) (containing 1 × 10⁸ ufc) was added per well, and plates incubated during 1 h at 37°C. Plates were then washed three times with washing buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20) for removal of unbound bacteria, and fluorescence signal quantified with the help of a microplate fluorescence reader (Synergy H1, BioTek, CA, United States). The control samples for each strain included wells treated with each human protein, but not incubated with FITC-labeled bacteria, and wells treated with BSA followed by the incubation with the bacterial suspensions.