Fm 1 43fx

A total volume of 200 µL was prepared for each sample, and measured in a 8-well coverglass chambers (Nunc Lab-Tek) at 20 °C. The samples were prepared in triplicates, and the average and standard deviation shown. Biofilms of the extensively resistant P. aeruginosa isolates 2978, 5550, 5832 and 5842 were established by culturing for 24 h in 8-well coverglass chambers (Nunc Lab-Tek) filled with LB medium containing 1% D-Glucose, starting from an overnight culture diluted 1:100. The medium was removed and subsequently treated with 45.2 µg P[5]a or PBS for an additional 16 h, as previously reported^{59 (link)}. The biofilms were then washed with water, and FM1-43FX (Invitrogen) was added to stain the bacterial cell membrane. The biofilm was fixed in 16% paraformaldehyde, 2.5% glutaraldehyde and 4.0% acetic acid in 0.1 M phosphate buffer (pH 7.4) overnight at 4 °C. Biofilms were visualized using an Axio Observer Z1 microscope (Carl Zeiss, Jena, Germany). Green fluorescent signal was obtained using excitation light at 470 nm, while collecting the emitted light of 515–535 nm.

The presence of functional mechanosensitive channels was confirmed using a FM1-43FX dye uptake assay. Organoids were incubated in Advanced DMEM/F12 containing FM1-43FX (5 Μm final concentration; Invitrogen) for 1 min and then washed three times in Advanced DMEM/F12. Organoids were dissociated to single cells by 40 min incubation in Accutase then added Advanced DMEM/F12 to inactivate Accutase. The cells were then plated onto slides using the Cytospin method. For nuclear staining, Hoechst dye was used. The cells were imaged to confirm dye uptake and immediately fixed with 4% paraformaldehyde. For some experiments, cells were fixed and stained with otic lineage associated antibodies to confirm the identity of hair cells. Cells were examined using Nikon Ti Eclipse confocal microscope. Fields were chosen at random.

For staining of functional HCs within the neuromasts, the vital dye FM1-43FX (Invitrogen, F-35355)—which enters mature HCs through mechanotransduction-dependent activity—was applied at a concentration of 3 μM to live 5 dpf larvae for 45 s in the dark. After quickly rinsing three times with fresh egg water, the larvae were anesthetized in 0.02% MS-222 and fixed with 4% PFA in PBS for 2 h at room temperature or overnight at 4°C.