Tototm 3 iodide

Control shLacZ JW23.3, shTyk2 #1 JW23.3, shTyk2 #2 JW23.3, and shTyk2 #3 JW23.3, were cultured in standard conditions with 2 µg/mL puromycin, then plated at 5000 cells/well in 96‐well plates. For proliferation assays, cells were plated in phenol‐red free DMEM with 10% FBS. Cells were imaged every hour for 48 hours using the IncuCyte FLR imaging system (Essen Bioscience, Ann Arbor, MI, USA) and analyzed for quantitation using IncuCyte ZOOM Analysis Software (Essen Bioscience, Ann Arbor, MI) Phase images were used to determine percent confluence and subsequent wells were normalized to initial confluence. For cell death assays, 50nM TOTO^TM‐3 iodide (ThermoFisher Scientific, USA) was added to the phenol‐red free media with reduced serum as a stress (5% FBS). Cells were imaged every hour for 48 hours using the IncuCyte FLR imaging system (Essen Bioscience, Ann Arbor, MI). For quantification of cell death, the TOTO^TM‐3 iodide fluorescence was normalized to the confluency factor calculated from the phase of each respective well.

To prepare the samples, inocula were prepared as previously described with an adjusted OD of 0.01, and 500 μl were distributed in 24-well plates on a Thermanox^TM coverslip placed at the bottom of 24-well plates (cell culture treated side up). After 24 h of incubation, the coverslips were washed twice in PBS and stained with SYTO^TM 9 at 1 μM and (i) propidium iodide (Thermo Fisher Scientific, Waltham, MA, United States) at 20 μM to label live and damaged or “dead” bacteria, (ii) SYPRO^® Ruby (Thermo Fisher Scientific, Waltham, MA, United States) (v/v) to label proteins, or (iii) wheat germ agglutinin (WGA) associated with the Alexa Fluor^TM 350 conjugate (Thermo Fisher Scientific, Waltham, MA, United States) at 100 μg/ml to label PIA with TOTO^TM-3 iodide (Thermo Fisher Scientific, Waltham, MA, United States) at 2 μM to label extracellular DNAs. Each label was diluted in 0.9% NaCl. After 30 min of incubation in the dark at room temperature, each coverslip was washed two times with PBS and placed in a 24-well Krystal plate with glass bottom (Dutscher, Porvair, United Kingdom) with the biofilm-side lower before observation with confocal laser scanning microscopy (CLSM) (LSM 710 NLO, Zeiss, Germany). Fluorochromes-labeled matrix compounds were imaged and their volume quantified using IMARIS software (Imaris, RRID:SCR_007370).