Vectashield

The hMSCs were imaged in 25% v/v VectaShield (Vector Laboratories) in glycerol (Sigma)^{58 (link)}. First a drop of the 25% v/v VectaShield in glycerol was added to the cells and a 7 mm diameter glass coverslip (VWR) placed on top to reduce oxygen exchange. The lid of the 8 well glass bottom μ-slide was also replaced to further reduce available oxygen. Imaging was carried out on a Zeiss Elyra PS.1 AxioObserver Z1 motorized inverted microscope with an electron multiplying charge-coupled device (EMCCD) camera (Andor iXon DU 897), an alpha Plan-Apochromat 100x/1.46 NA immersion oil DIC VIS Elyra objective and a 640 nm solid-state laser (150 mW). ZEN black image software v.2012 (https://www.zeiss.com/microscopy/us/products/microscope-software.html) was utilized to acquire the movies. Images were captured with EPI illumination, in ultra-high power mode (PALM_uHP), in 16-Bit depth with a pixel size of 100 nm and an image size of 24.8 μm × 24.8 μm. AlexaFluor647 was excited at 640 nm with an exposure time of 50 ms per frame at 80% laser power with an EMCCD gain of 10% and the fluorescence emission was acquired over 15,000 frames. As hMSCs are much bigger than the field of view with a 100 × objective, images were taken with as much of the cell in as possible.

Human CAFs were plated to confluence on 12 mm glass coverslips in DMEM supplemented with 10% FBS and 50 µg/ml ascorbic acid, and media was changed daily for 7 days. Cells were then lysed on day 7 (25 mmol/L Tris-HCl, pH 7.4; 150 mmol/L sodium chloride; 0.5% Triton X-100; and 20 mmol/L ammonia hydroxide) for 3–5 min. Cellular debris was carefully washed away with 1X PBS. Resultant cell free ECMs were fixed in 4% paraformaldehyde for 15 min at room temperature and then blocked with 5% FBS in 1X PBS. ECMs were then incubated in mouse anti-fibronectin antibody (diluted 1:100, BD Biosciences) overnight at four degrees, washed twice, and then incubated in goat anti-mouse AlexaFluor 488 secondary (diluted 1:500, Life Technologies), washed four times, mounted in Vectashield (VWR, 101098–044), and sealed with nail polish. Immunofluorescence was analyzed on a confocal microscope (LSM 700; Carl Zeiss, Jena, Germany) at room temperature with Zen 2009 software. ImageJ was used to adjust brightness and contrast.

Exponentially growing cultures (37 °C, 230 r.p.m.) of indicated strains were harvested at OD₆₀₀=0.6, diluted to 10⁵ CFU/ml in LB w/o salt, dispensed (8 ml aliquots) in six-well plates (Sarstedt, Numbrecht, Germany) with inclined glass coverslips (24×24 mm, VWR International, Stockholm, Sweden), and incubated for 48 h at 28 °C to allow biofilm formation. Removed coverslips were washed twice in PBS before and after 1 h fixation in 4% formaldehyde, then immersed in 3 μmol/l h-HTAA or h-FTAA in PBS. Following 30 min incubation in the dark, coverslips were washed twice in PBS, then prepared for microscopy analysis. In negative controls, coverslips were immersed in PBS without added LCOs. Biofilm formed on the surface at the air–liquid interface was analysed on coverslips immobilised (Vectashield, VWR International) onto glass slides. The 20× dry objective on an Olympus FV1000 confocal microscope (Olympus, Stockholm, Sweden) was used with preset detection settings for transmission, GFP (h-HTAA) and Cy3 (h-FTAA) fluorescence.