Bovine serum albumin solution

The ability of the strain YPG14 to bind to mucin was evaluated as previously described with appropriate modifications [31 (link)]. The 100 μL of mucin at a concentration of 10 mg/mL were added to a 96-well plate and incubated overnight at 4 °C. The plate was washed twice with PBS and saturated with a 2% (w/v) bovine serum albumin (BSA) solution (Sigma-Aldrich, St Louis, MO, USA) for 4 h at 4 °C. The plate was washed twice with PBS, and then 100 μL YPG14 bacterial suspension was added to each well. The plate was incubated at 37 °C for 1 h. After incubation, the wells were washed 4 times with PBS to remove non-adherent bacteria. The 200 μL of 0.5 % Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) was added to each well and incubated for 2 h at room temperature, then the wells were scraped with a sterile tip and the suspension was taken for tenfold serial dilutions. Then, 100 µL of the dilutions was plated on the MRS agar plates and incubated overnight at 37 °C. Colonies were counted and the CFU per mL was calculated.

For immunofluorescence analyses, the fixed oral mucosa equivalents were incubated in 0.2% Triton-X (Sigma-Aldrich, St. Louis, MO, USA) at room temperature (RT) for 20 min. Afterwards, the tissue equivalents were washed with DPBS three times for 10 min at 300 rpm on a thermoshaker (Cellmedia, Carl Roth, Karlsruhe, Germany). This was followed by incubation with CD31 antibody (monoclonal mouse anti-human, clone JC70A, Dako, Santa Clara, CA, USA, 1:50 in 1% Bovine Serum Albumin (BSA) solution (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at RT and afterwards overnight at 4 °C on the thermoshaker at 300 rpm. The next day, the tissue equivalents were washed with DPBS three times for 10 min and incubated with the secondary antibody Alexa Fluor^® 488 (polyclonal goat anti-mouse, ThermoFisher Scientific, Waltham, MA, USA, 1:200 in 1% BSA solution) for 2 h at RT on the thermoshaker at 300 rpm. The tissue equivalents were then washed three times with DPBS, and vascular-like structures were analyzed with confocal fluorescence microscopy (TCS SP8, (Leica Microsystems, Wetzlar, Germany)).

DMEM/F12, fetal bovine serum (FBS), penicillin, streptomycin, amphotericin B, l-glutamine solution and trypsin–EDTA solution C were purchased from Biological Industries (Beit Haemek, Israel). Bovine insulin, hydrocortisone, ovine prolactin, bovine serum albumin (BSA) solution, hyaluronidase, DNase I and heparin were purchased from Sigma Aldrich Israel (Rehovot, Israel). Collagenase type II was purchased from Worthington Biochemical Corporation (Lakewood, NJ, USA).
For lipid extraction, methanol and chloroform (both analytical reagent grade) were purchased from Bio-Lab (Jerusalem, Israel). For HPLC analysis, chloroform and ethanol (used at 97:3 v/v, both analytical reagent grade) and methanol (HPLC grade) were purchased from Bio-Lab. TG (triglyceride triolein, purity > 99%) was purchased from Supelco (Bellefonte, PA, USA). Phospholipid standards were from Sigma Aldrich Israel and consisted of the following: PE (1,2-dioleoyl-sn-glycero-3 phosphoethanolamine, 10 mg phospholipid per mL CHCl₃, purity 99%), PI (l-α phosphatidylinositol ammonium salt, from bovine liver, purity 98%), PS (1,2-dioleoyl-sn-glycerol-3-phospho-l-serine sodium salt, purity 95%), PC (1,2-dioleoyl-sn-glycero-3-phosphocholine, purity ≥ 99%) and SM (sphingomyelin; from bovine brain, purity 97%). Commercial standard mixes of phospholipids and TG were injected to determine retention times.

Cells in the hydrogel were fixed at room temperature in 10% formalin for 30 min. After 3 times of PBS washes, cells were permeabilized using a 0.1% Triton X-100 solution for 5 min. Hydrogels were washed 3 times with PBS, blocked using a 1% bovine serum albumin (BSA) solution (Sigma) supplemented with 1% goat IgG (Jackson ImmunoResearch), and the blocking solution was maintained for 1 h. In some cases, samples were stained for a specific molecular endpoint, but in all cases, DAPI nuclear stain was applied for profiling colony organization. Monocolonal rabbit anti human primary antibodies (e.g., ERBB2, β-catenin, β4-integrin) (Abcam) were diluted in blocking solution (1:500), incubated for 1 h, and followed by 3 PBS washes. Goat anti rabbit secondary antibody conjugated with Alexa 488 and Alexa 565 (Life Science Technology) was diluted in the blocking solution (1:250), conjugated to primary antibody for 1 h, and followed by 3 PBS washes. Subsequently, nuclei were stained with 4′-6-diamidino-2-phenylindole (50 ng/mL) (DAPI, Life Science Technology).

This study utilized PSF hollow fiber (PSF HF) membranes supplied by Polymem (Fort Worth, TX, USA) as the substrates for atomic layer deposition (ALD). The membranes underwent chlorine washing and glycerin conditioning steps before the deposition process. The following materials were employed for the ALD process: titanium (IV) chloride (TiCl₄, 99.9%, CAS: 7550-45-0), diethylzinc (DEZ, (C₂H₅)2Zn, >95%, CAS: 93-3030), trimethylaluminum (TMA, Al(CH₃)₃, >98%, CAS: 93-1360), and deionized water. Argon gas, which was purchased from Linde (Dublin, Ireland), was utilized as received. Phosphate-buffered saline (PBS) from Roth (Karlsruhe, Germany) and bovine serum albumin (BSA) solution with a molecular weight of 67 kDa and a purity of ≥96% from Sigma-Aldrich (St. Louis, MO, USA) were used to evaluate the fouling resistance of both the untreated and modified PSF HF membranes. Deionized water from Millipore Milli-Q (Burlington, MA, USA) was used in all aqueous solutions throughout the experiment.