Syto60

Biofilms were grown under the conditions described above. For static biofilms ibidi^® μ-Plate 96 Well Uncoated plates were used instead of polystyrene microtiter plates. After 24 h, medium was removed gently by aspiration, and biofilms washed three times with PBS. Biofilms were stained as described previously (Okshevsky and Meyer, 2014 (link)) in PBS containing 10 μM Syto 60^® (Thermo Scientific), a red-fluorescent, membrane permeable dye staining live bacteria and 2 μM TOTO-1^® (Thermo Scientific), i.e., a green-fluorescent dye staining eDNA or DNA of bacteria with a compromised membrane. Imaging was performed on a Zeiss LSM700 confocal laser scanning microscope (CLSM) equipped with 555 and 635 nm lasers and a variable dichroic beam splitter for simultaneous recording of the emitted light from the two fluorophores by separate photomultipliers. All images were captured with a 63× objective and analyzed using Zen 2012 software (Zeiss).

The following lectins were included in the FLBA. A. naeslundii: Bananas musa acuminate (BanLec-FITC), Concanavalin A (ConA-FITC), Vicia graminea (VGA-FITC), and Triticum vulgare (WGA-FITC). L. paracasei subsp. paracasei: Agaricus bisporus (ABA-FITC) and Helix pomatia (HPA-FITC). S. epidermidis: WGA. S. mitis: VGA and WGA. Bacterial suspensions were washed twice with sterile PBS (4,696 g for 5 min), adjusted to an OD₅₅₀ of 0.1, and immobilized for 15 min on microscopy slides (SuperFrost Ultra Plus; Thermo Fisher Scientific) or in plasma-treated flow cells (µ-slide VI ibitreat; ibidi, Planegg/Martinsried, Germany). After rinsing with PBS, cells were stained with the respective lectins, incubated in the dark for 30 min, and rinsed with PBS to remove unbound lectins. The effect of OPN on lectin binding was tested by adding OPN to a concentration of 460 µM during lectin binding followed by washing with PBS or PBS with 460 µM of OPN. After lectin staining, bacterial cells were counterstained with SYTO® 60 (10 µM; Thermo Fisher Scientific).

THP1 monocytes were differentiated in 100 ng/mL TPA complete medium for 48 h and allowed to recover for 24 h on transwell inserts. Respective siRNA knockdowns were performed on iCECS (iCEC_ANGPTL4, iCEC_TTP and iCEC_Ctrl). Inserts containing differentiated THP1 cells were then introduced to the transfected iCECs and exposed to various pro- and anti- inflammatory stimuli for 10 h (Fig. 3e). Inserts were then washed with PBS twice and fixed in 1% glutaraldehyde for 10 min, rinsed with PBS and stained with SYTO 60 (Thermo Fisher, USA) for 30 min. Cotton buds were used to remove all unmigrated cells trapped in the upper chamber of the inserts. Inserts were rinsed again in PBS before the quantification of fluorescence using the CLx scanner and Image Studio V2.1 (LI-COR Biosciences, USA). Relative fluorescence as a readout for cellular migration was calculated by normalizing the intensities between test wells (THP1 and iCECs) and control wells (THP1 without iCECs).

To investigate the binding of OPN to bacterial cell surfaces, the protein was fluorescently labeled. OPN was dissolved in 3 mL of NaHCO₃ buffer (50 mM; pH 9.5) at room temperature, and fluorescein isothiocyanate (FITC; Sigma–Aldrich, Brøndby, Denmark) in dimethyl sulfoxide was added dropwise within 30 min, yielding a final molar OPN/FITC ratio of 1/10. After magnetic stirring for 5 h, the labeled protein was purified by dialysis for 48 h (3 mL against 1,000 mL) in a dialysis tube with a molecular weight cutoff of 3.5 kDa (Spectra/Por® RC; Spectrum Labs, Rancho Dominguez, CA). Then, labeled OPN was freeze-dried for 48 h (Triad cascade benchtop freeze dry system; Labconco Corp., Kansas City, MO). Bacteria were washed in phosphate-buffered saline (PBS; 4,696 g), adjusted to an OD₅₅₀ of 0.1, incubated with labeled OPN (20 µM/L) at 35°C for 15 min, washed twice with PBS to remove unbound protein, and counterstained with SYTO® 60 (10 µM; Thermo Fisher Scientific, Naerum, Denmark). A confocal microscope (Zeiss LSM 700; Carl Zeiss, Jena, Germany) equipped with a 63× oil immersion objective (alpha Plan-Apochromat; Carl Zeiss) was used for image acquisition. FITC and SYTO® 60 were excited at 488 and 639 nm and detected with 640 nm short- and long-pass filters, respectively.

Cells were seeded in either 96-well or 384-well microplates in RPMI-1640 or DMEM/F12. The optimal cell number for each cell line was determined to ensure that each was in growth phase at the end of the assay (~70% confluency). Adherent cell lines in the screens were plated 1 day prior to treatment with each compound using liquid handling robotics and assayed after 6 days of treatment with either the single agent or in combination with rTRAIL. Cells were fixed in 4% formaldehyde for 30 min and then stained with 1 μM of the fluorescent nucleic acid stain Syto 60 (Thermo Fisher Scientific, UK) for 1 hr. Quantitation of fluorescent signal intensity was performed using a fluorescent plate reader at excitation and emission wavelengths of 630/695 nm. The sensitivity of each cell line to various concentrations of compound was calculated as the fraction of viable cells relative to DMSO-treated cells following a 6 day exposure. All screening plates were subjected to stringent quality control measures and a Z-factor score comparing negative and positive control wells was calculated across all screening plates (median = 0.70, upper quartile = 0.86, lower quartile = 0.47, n = 4857 plates).