Glass slides

Biofilms were grown in flow-chambers with individual channel dimensions of 1 x 4 x 40 mm as described previously [65 (link)]. One hour after inoculation, with bacteria, the growth medium flow (0.2 mm/s corresponding to laminar flow with a Reynolds number of 0.02) was started. When required, eDNA in biofilms was stained with 1 μM ethidium bromide prior to microscopy. Tobramycin (10 μM) was added to the biofilm medium after 4 days of cultivation. After 24 h of tobramycin treatment, propidium iodide (10 μM) was added to the flow cells to visualize the dead cells via confocal laser scanning microscopy. P. aeruginosa PA14 and petA mutant biofilms were grown under static conditions over 48 h at 37°C on glass slides (Ibidi) incorporating 300 μl chambers. After 48 h incubation, spent medium was removed and the biofilm eDNA stained with YoYo-1 (40 μM).

For immunofluorescence experiments, HKs were cultured directly on glass slides (Ibidi) until confluent, as described above. Live cells were labeled with monoclonal antibodies at a concentration of 10 μg/ml for 30 min at 4°C. Cells were then fixed on ice using 3.7% paraformaldehyde for 10 min followed by permeabilization in 0.1% triton for 10 min at room temperature. Goat anti-human IgG cross-adsorbed secondary (Life Technologies) was then used prior to mounting with prolong gold containing Dapi (Life Technologies). Widefield images were acquired using a DMRXA2 microscope (Leica, Wetzler, Germany) equipped with a 63X/1.40 NA oil immersion objective. Images were acquired with an ORCA digital camera (Hamamatsu Photonics, Bridgewater, NJ). Analysis was preformed using Fiji ImageJ.

Glass slides (Ibidi, Germany) were washed with boiling piranha solution (70% of pure sulfuric acid with 30% of a 30% hydrogen peroxide solution) for 30 min. After intensive washing with PBS, Glass slides were functionalized with 5 μg/ml of anti-CD3 (OKT3) (eBiosciences, Thermo Fisher Scientific) or 5 μg/ml of IgG or 100 μg/ml of poly-L-lysine for 30 min at room temperature followed by extensive rinsing with PBS + 0.1% of BSA. A total of 8 × 10⁵ electroporated T cells were washed and resuspended in PBS + 0.1% of BSA, then incubated on each substrate and fixed with 4% of paraformaldehyde during 20 min (Sigma, Germany) after 2, 4, 8, or 16 min after incubation. Cells were washed with PBS + 0.1% of BSA and incubated in 50 mM of NH₄Cl (Sigma, Germany) for 20 min and then rinsed again with PBS + 0.1% of BSA. Cells were permeabilized with 0.5% of Triton-X 100 in PBS + 0.1% of BSA during 15 min before being rinsed with PBS + 0.1% of BSA. Cells were then labeled with 0.1 μg/ml of Phospho-ZAP70/Syk (Tyr319, Tyr352) Monoclonal Antibody (n3kobu5) PE (eBioscience, Thermo Fisher Scientific) in PBS + 0.1% of BSA + 0.05% of saponin (Sigma, Germany) during 1 h. After extensive rinsing with PBS, cells were imaged using a confocal microscope (Zeiss LSM 880 AiryScan) with a 63× 1.4 NA objective.