Glutaraldehyde

Zwittergent 3-14 detergent (code: 693017) was purchased from Millipore (Burlington VT, USA), agarose (code: BY-R0100) was from BIOWEST (Nuaillé, France), Freund’s adjuvant (code: P5881) and glutaraldehyde (code: G6257) were purchased from Sigma (Tokyo, Japan), Coomassie brilliant blue (code: 6104-59-2), methanol, and acetic acid were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The lysis buffer (PBS containing 1% Zwittergent), staining solution (0.2% (w/v) Coomassie brilliant blue, 5% (v/v) methanol, 1% (v/v) acetic acid in distilled water), and destaining solution (40% (v/v) methanol, 10% (v/v) acetic acid in distilled water) were prepared in the lab.

For SEM observation, dual-species biofilms were grown on silicone
coupons following a structured protocol. Initially, samples were washed
with phosphate-buffered saline (PBS) at pH 7.2, prepared using KH₂PO₄ (Exodus Science, Brazil) at a concentration
of 10 mM, NaCl (Dinamica, Brazil) at 137 mM, and KCl (LabSynth Ltd.a.,
Brazil) at 2.7 mM. After washing, biofilms were fixed with 3% glutaraldehyde
(Sigma-Aldrich, Portugal) in PBS for 1 h to preserve cellular structure.
Subsequently, samples underwent a dehydration process using an ethanol
gradient of 10%, 30%, 70%, and 100% ethanol solutions, with each step
lasting 15 min. This gradient ensures complete dehydration to avoid
structural distortion during SEM observation. After fixation and dehydration,
the coupons were dried in a laminar flow chamber at room temperature
(22 ± 2 °C) for 24 h to ensure complete removal of solvents.^{53 (link),78 (link)} After drying, the samples were coated with a gold film (120 nm thick)
using a metallizer (Quorum Q 150R ES, England) for 12 min, a critical
step to increase electronic conductivity during imaging. The morphology
and interactions of the biofilms were then observed using SEM/EDX
(Tescan VEGA3, Czech Republic) under vacuum conditions with an accelerating
voltage of 10 kV.

A custom strain device was manufactured from stainless steel (Figure S3), and cells were grown on modified stretchable PDMS surfaces. PDMS sheets (0.25 mm thick), cut into 5 by 1.7 cm rectangles, and PDMS rings, 2 mm thick with 1.0-cm inner and 1.6-cm outer diameters, were cleaned in PCC-54, ethanol, and finally water. After drying, PDMS rings were placed in the middle of the PDMS sheets, and the PDMS was treated with air plasma (PDC-32G, Harrick Scientific, http://www.harricksci.com/) at 250 mbar for 30 s to render the surface hydrophilic. For noncovalent attachment of Fn-u to the surface, plasma-treated surfaces were used. For covalent attachment of Fn-u, amino groups were attached to the plasma-treated surface with 3-aminopropyltriethoxysilane vapor in an evacuated chamber for 1 h. Vapor-based silanization resulted in significantly lower surface fluorescence than solution-based silanization (data not shown), as has been previously reported [50 (link)]. After silanization, 0.125% glutaraldehyde (Sigma-Aldrich) was added and the sample incubated for 20 min. Finally, the surface was rinsed with water and dried prior to assembly of the strain device.
All metal strain device parts were autoclaved prior to assembly within a tissue culture hood. PDMS sheets were clamped into the strain device, and the culture surface within the PDMS ring was incubated with 0.03 g/l Fn-u for 1 h. Unreacted glutaraldehyde was quenched by incubation with DMEM plus 10% NCS for 20 min at 37 °C. Then 20 × 10³ fibroblast cells/cm² were seeded onto the PDMS surface and allowed to adhere for 30 min prior to removal of unbound cells and replacement with DMEM plus 10% NCS, 0.005 g/l Fn-DA, and 0.045 g/l Fn-u. After 24 h, cells were washed two times in 37 °C PBS and extracted, leaving behind the detergent-insoluble Fn matrix, with a 5-min incubation in 0.5% Triton X-100 plus 20 mM NH₄OH in PBS (pH ∼9.7). The strain device with attached cell-free matrix was then imaged. Three separate fibril detachment experiments were performed with both Fn labeling approaches, while four and six separate stretching experiments were performed with amine/cys Fn-DA or cys/cys Fn-DA, respectively, on Fn-u that was covalently attached to the membrane. During strain application, it was found that mounting the PDMS ring on the PDMS membrane prior to treatment with air plasma most often resulted in the PDMS ring sliding on the membrane surface during stretching. Strain samples were discarded if the ring stuck to the membrane, thus leading to inhomogeneous strain application, or leaked culture medium.