C10600

A microscopic adhesion assay was used to assess bacterial adhesion on Cn substrates. Substrates were incubated for 2 h in 200-µl bacterial suspensions adjusted in PBS to a concentration of 10⁹ cells ml⁻¹. After 2 h, the substrates were gently rinsed by 3 consecutives washing in PBS and directly imaged using an inverted optical microscope (Zeiss Axio Observer Z1) equipped with a model C10600 Hamamatsu camera.
A crystal violet assay was also used. Microtiter wells were coated overnight at 4°C with 1 µg/well collagen type II–0.1 M sodium carbonate (pH 9.5). The plates were washed with 0.5% (vol/vol) PBS with Tween 20 (PBST). To block additional protein-binding sites, the wells were treated for 1 h at 22°C with 2% (vol/vol) bovine serum albumin (BSA)–phosphate-buffered saline (PBS). The wells were then incubated for 2 h at 37°C with 1 × 10⁸S. aureus Phillips cells–PBS. After washing with PBS was performed, adhering cells were fixed with 2.5% formaldehyde for 30 min and stained with 1% crystal violet for 1 min. After washing, 100 µl of 10% acetic acid was added, and absorbance at 595 nm was recorded using an ELISA plate reader (BioRad).

Target cells (MATα) were immobilized mechanically into porous membranes. One milliliter of undiluted cell suspension was filtered through 5-μm pore size polycarbonate membrane (it4ip, Belgium). The filter was then rinsed with phosphate-buffered saline (PBS) buffer, carefully cut (~0.5 cm × 0.5 cm), and stuck with double face adhesive tape on one side of the microscope Petri dish while avoiding dewetting. On the other side, 50 µL of the opposite mating type diluted 100× suspension (MATa) were dropped and let to settle for 5 min before being rinsed in PBS. Cell–cell force spectroscopic experiments were then performed at room temperature (20 °C) in filtered PBS buffer, using a JPK Nanowizard 4 atomic force microscope combined with an inverted optical microscope (Zeiss Axio Observer Z1 equipped with a Hamamatsu camera C10600, Zeiss AG) and connected to a pressure pump unit and a pressure controller through a microfluidic tubing system (Cytosurge AG).