μ slide

The lytic effect of phage on the prevention of biofilm formation and its eradication was evaluated by CLSM. Brieflyan overnight bacterial culture (diluted 1:100) was distributed into an 8-well μ-Slide (Ibidi) to form biofilm. For prevention experiments, bacteria were simultaneously incubated (at 37°C for 24 h) in the presence of different titres of phages. For eradication experiments, bacteria were first let form biofilm into an 8-well μ-Slide (Ibidi) for 24 h at 37°C, and then treated with different phage titers. Bacteria viability and biofilm thickness after phage co-incubation/treatment was determined by CLSM after staining cells with Syto9 (488 nm/500–540 nm) and propidium iodide (PI) (561 nm/600–650 nm) as recommended by the manufacturer (Live/dead BacLight Bacterial Viability Kit Molecular Probes, Life technologies). Samples were analyzed by the microscope TCS SP5 (Leica, Heidelberg, Germany) using a 63× objective and a pinhole aperture of 1.0 Airy. For each image, the mean of fluorescent intensity was calculated as previously described.

HEK-293 cells stably expressing the M₃ muscarinic acetylcholine receptor were cultured in Dulbecco’s modified Eagle’s medium (DMEM/F12) supplemented with 10% fetal calf serum, 100 U/mL penicillin G, and 0.1 mg/mL streptomycin as described [23 (link)]. Stock cultures of HEK-293 cells were grown in the presence of 0.5 mg/mL G418. Mouse embryonic fibroblasts (MEFs) from CIB1-deficient mice were made by J.L. Black in the laboratory of Dr. Leslie V. Parise (University of North Carolina at Chapel Hill, NC, USA) [36 (link),37 (link)]. These MEFs, along with MEFs from Gα_q/11 double-deficient mice [38 (link)], were cultured in DMEM/F12 medium with 10% fetal calf serum, 100 U/mL penicillin G, and 0.1 mg/mL streptomycin. Transfection of HEK-293 cells was performed with Lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany), while MEFs were transfected with Turbofect (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions. For microscopy, the cells were seeded onto poly-l-lysine-coated 8-well slides (μ-slide; ibidi GmbH, Martinsried, Germany). Before experiments, the cells were kept in serum-free medium overnight.

Collagen-I gel matrix was prepared as described previously (Mardakheh et al., 2015 (link)), with slight modifications. Briefly, 5x DMEM adjusted with 0.1M NaOH and 3.7% NaHCO₃ was mixed with pepsinized bovine collagen-I (Advanced BioMatrix) and diluted with dH₂O to 1.7 mg/ml of Collagen-I whilst on ice. The mixture was then poured into individual wells of iBidi μ-Slide with 18 wells and allowed to set at 37°C for 2 hrs. Subsequently, the cells were plated on the top of the set matrix in complete media. After 2 days of cell invasion through the collagen-I gels, cultures were fixed with 10% Neutral Buffered Formalin (NBF) for 30 min before further processing for dual RNA-FISH and antibody staining, and imaging by confocal microscopy.