Glass bottom 24 well plate

Cells were grown in 35 mm dishes, glass bottom 24-well plates (MatTek), or 8-well chamber cover glasses (Lab-Tek). Infected cells were imaged after replacing the culture media with phenol red-free imaging medium (Fluorobrite, Gibco) containing 20 μM DFHBI-1T (Lucerna), 25 mM HEPES (Gibco) at 37 °C for 30 min. The imaging medium plus 1% FBS, 1% PenStrep, and 2 mM glutamine was used for time-lapse imaging. Cell viability was assessed with 3 μM propidium iodide (Invitrogen). Autofluorescence was assessed relative to SINV TE (no aptamer)-infected cells and was minimized by adjusting the laser power and gain in each experiment. A laser scanning confocal microscope (Zeiss LSM780FCS, Axio Observer Z1, Zen Software) with 488 nm excitation was used to activate the Broccoli-DFHBI-1T complex. In dAP7 cells, the true signal was differentiated from autofluorescence with the lambda scan that captured an emission spectrum in each pixel. The Broccoli-DFHBI-1T signal had an emission peak at 507 nm and appeared green in lambda mode, while the autofluorescence signal had no peak, extended from green to far red in its spectrum, and appeared yellow. In time-lapse experiments, temperature and CO₂ were kept at the same level as the culture condition for each cell type with a heated chamber at 24% humidity. ImageJ Software was used for image analysis.

Cells were seeded on glass-bottom 24-well plates (MatTek). Where indicated, cells were seeded in glass-bottom plates covered with 10 µg ml⁻¹ fibronectin. For analysis of 3D morphology, cells were seeded on top of 2.3 mg ml⁻¹ collagen-I gel over a glass-bottom dish (MatTek) in medium and allowed to adhere for 24 h. Cells were fixed in 4% PFA and permeabilized in PBS with 0.2% Triton X-100. Where indicated (i.e., septin staining) cells were fixed in ice-cold methanol for 15 min and permeabilized in PBS with 0.2% Triton X-100. Samples were blocked in 3% BSA with 0.1% PBS Tween (PBST) for 3 h. Primary antibodies (Table S3) were diluted in 3% BSA in PBST for 2 h. Wells were then washed three times for 10 min each in 3% BSA PBST, followed by the addition of the appropriate secondary (Alexa Fluor; Invitrogen). After three 15-min washes in PBS, samples were mounted and analyzed.

Cells were grown in 35 mm dishes, glass bottom 24-well plates (MatTek), or 8-well chamber cover glasses (Lab-Tek). Infected cells were imaged 30 min after replacing the culture media with phenol-red-free imaging medium (Fluorobrite, Gibco) containing 20 μM DFHBI-1T (Lucerna), 25 mM HEPES (Gibco) at 37 °C, 1% FBS, 1% PS and 2 mM glutamine. Autofluorescence was assessed relative to SINV TE (no aptamer)-infected cells and was minimized by adjusting the laser power and gain in each experiment. The laser scanning confocal microscope (Zeiss LSM780FCS, Axio Observer Z1, Zen Software) with A 488 nm laser excitation was used to activate the Broccoli-DFHBI-1T complex. In dAP7 cells, the true signal was differentiated from autofluorescence with the lambda scan that captured an emission spectrum in each pixel. The Broccoli-DFHBI-1T signal had an emission peak at 507 nm and appeared green in lambda mode, while the autofluorescence signal had no peak, extended from green to far red in its spectrum, and appeared yellow. In time-lapse experiments, temperature and CO₂ were kept at the same level as the culture condition for each cell type with a heated chamber at 24% humidity. ImageJ Software was used for image analysis.

For microscopy analysis, P. aeruginosa biofilms were grown in glass-bottom, 24-well plates (MatTek Corporation, Ashland MA, USA) as described above. After 7 h incubation including 1 h treatment, biofilms were rinsed twice with PBS before being stained with LIVE/DEAD® BacLight™ bacterial viability kit reagents (L-7007, Molecular Probes) according to the manufacturer's procedure. One microliter of each of the two components were mixed thoroughly in 1 mL of PBS, then 0.3 mL of this solution was trapped between the sample and the glass microscopy slide and allowed to incubate at room temperature in the dark for 20 min. The samples were observed with an Olympus FV1000 Confocal Inverted Microscope, and imaged with a Leica DFC 480 camera. Cells that were stained green were considered to be viable, those that stained red and stained both green and red were considered to be non-viable.

In order to identify and localize individual bacteria in the dual-species biofilms, cocolonized biofilms were prepared on a fixed epithelial substratum as described above, except we used glass-bottom 24-well plates (MatTek Corp., Ashland, MA) and chromosomally encoded fluorescent constructs (EF3030-GFP [green] and UAMS-1–dsRED [red]). Image stacks were acquired by CLSM at 630× magnification on a Zeiss Axiovert 200 M inverted microscope with an attached Zeiss LSM 510 Meta NLO imaging system. Images were produced from the raw LSM files by using the freely available ImageJ software analysis program.