Dextran sulphate

A 10⁷ CFU mL⁻¹C. sakazakii ATCC 25944 suspension was prepared and placed on a microscope slide as described above. The fluorescent in situ hybridization was performed in three main steps: (i) sample fixation/permeabilization, (ii) hybridization and (iii) washing of the unbound probe. The dried 20 μL samples were covered with 30 μL of 4% (wt/vol) paraformaldehyde (Sigma), followed by 50% (vol/vol) ethanol (Fisher Scientific) for 10 min each, and subsequently air dried. The samples were then covered with 30 μL of hybridization solution containing 10% (wt/vol) dextran sulphate (Sigma), 10 mM NaCl (Sigma), 30% (vol/vol) formamide (Sigma), 0.1% (wt/vol) sodium pyrophosphate (Sigma), 0.2% (wt/vol) polyvinylpyrrolidone (Sigma), 0.2% (wt/vol) ficoll (Sigma), 5 mM disodium EDTA (Sigma), 0.1% (vol/vol) Triton X-100 (Sigma), 50 mM Tris–HCl (pH 7.5; Sigma), and 200 nM of EUB338 probe (Panagene). Samples were covered with coverslips, placed in moist chambers, and incubated for 60 min at 61 °C. Subsequently, the coverslips were removed, and the slides were submerged in a prewarmed (61 °C) washing solution containing 15 mM NaCl (Sigma), 1% (vol/vol) Triton X-100 (Sigma), and 5 mM Tris base (pH 10; Sigma). Washing was performed at 61 °C for 30 min, and the slides were subsequently air dried. The samples could be stored (dark) for a minimum 24 h before observation, if needed.

Unless otherwise specified, media, medium supplements and reagents used for cell cultures were purchased from Thermo Fisher Scientific, (Waltham, MA, USA). The human RPE cell line ARPE19 (American Type Culture Collection, Manassas, VA, USA) was cultured in DMEM supplemented with 10% fetal calf serum (DMEM/FCS). To generate cell-derived extracellular matrix (ECM) coatings, ARPE19 cells were seeded at 80–90% confluence in DMEM/FCS (uncrowded conditions). Upon reaching confluence, the media was changed to DMEM/FCS supplemented with the macromolecular crowder dextran sulphate (200 μg/ml) and ascorbic acid (30 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA) (crowded conditions). Confluent ARPE19 monolayers were cultured for indicated times with half media changes twice per week. Wells were then decellularized by three successive incubations with 0.5% deoxycholate (Sigma) in distilled H₂O for 15 min at room temperature, followed by three successive washes with PBS to remove cell debris and residual detergents. Wells then were treated for 20 min with 10 U/ml of TURBO DNase (Thermo Fisher Scientific, Invitrogen) in PBS to remove residual DNA then washed three times in PBS.