Costar 3524

To evaluate the biofilm structure, the dual-species biofilms were formed in 24-well plates (Costar^® #3524, Corning Inc., Corning, NY, USA) and evaluated by scanning electron microscopy (SEM). In brief, dual-species biofilms of C. albicans and S. mutans were formed in 24-well plates and exposed to the experimental solutions, as previously described. After the last treatment, the wells were gently washed with NaCl (0.85%), and the biofilms were dehydrated using the following series of ethanol concentrations: 70% for 10 min, 95% for 10 min, and 100% for 20 min, followed by air drying for 20 min [41 (link)]. Next, the bottom of each well was cut with a flame-sterilized scalpel blade (number 11, Solidor, Lamedid Commercial and Services Ltd., Barueri, Brazil), and the biofilms were coated with gold and evaluated using an electron microscope (S-360 microscope, Leo, Cambridge, MA, USA).

24-well polystyrene plates (Costar 3524, Corning Incorporated) were coated with porcine mucin (10 mg/mL, Sigma) in distilled water (200 μL/well; 4 °C, overnight). After discarding the mucin solution, wells were washed twice with a 150 mM NaCl solution and 1 mL per well of fresh MRS, supplemented or not (vehicle: ethanol) with the indicated polyphenol (at concentrations ranging from 5 to 50 µM) and inoculated with 10⁷ colony forming units (CFUs)/mL of a culture in stationary phase of L. paracasei ATCC334 or L. rhamnosus GG strains. Plates were incubated at 37 °C for 24 h. Cells attached to the well walls were quantified as described previously [16 (link)]. After incubation, the medium was removed from each well, and the plates were washed twice in a 150 mM NaCl solution to remove loosely attached cells. We added 1 mL of a 150 mM NaCl solution to each well before repeated pipetting to detach the biofilm, and serial dilutions of biofilm recovered suspension were spotted onto MRS agar plates. Each strain and/or condition was tested in at least three independent experiments, each with three biological replicates.

The biofilm formation ability of AP2044 strain and lab-WT was determined by the crystal violet staining method and field-emission scanning electron microscope (FE-SEM) as described previously with minor adjustment. Briefly, the strains were cultured overnight and adjusted to a final OD₆₀₀ of 0.1. A total of 20 μL of each bacterial suspension and 180 μL of LB broth (Haibo, Qingdao, China) were inoculated into a 96-well polystyrene microtiter plate (Costar#3524, Corning, United States) in triplicate. After incubation at 37°C overnight, the plate was washed with phosphate-buffered saline (PBS, Solarbio, Beijing, China) to remove planktonic cells, and the plate was stained with crystal violet (Solarbio, Beijing, China) and solubilized with 95% ethanol (v/v), after which its absorbance was measured at 570 nm.
In addition, the biofilm formation capacity of each strain was used for SEM analysis. Biofilms were cultured for 24 h on coverslips as described above and planktonic cells were removed using PBS. First, the cells were fixed with 2.5% glutaraldehyde in PBS for 4 h. Second, the cells were gently washed with PBS twice. The samples were then gradually treated with ethanol (30, 50, 70, 80, 90, and 100%) for 20 min at each concentration. The dried samples were coated with platinum and visualized using FE-SEM (Thermo Fisher Scientific, Waltham, Massachusetts, United States).

The evaluation of endothelial cell migration is the most frequent method for studying chemotaxis in angiogenesis using transwell plates (Costar, #3524, Corning Incorporated, Waltham, NY, USA) with 8 μm diameter pores filters. Briefly, the bottom chambers were filled 600 μL with or without VEGF (25 ng/mL) and various concentrations of VH02. The insert filters were placed into the wells by sterile forceps. Then, cells 1 × 10⁴ cells/well were suspended in 100 μL of media and seeded in the upper chambers. The cultures were allowed to migrate at 37 °C for 16 h. After incubation, the chemoattractant medium in the bottom chambers was removed and replaced with 8 μM Calcein AM fluorescent dye in medium at 37 °C for 45 min. The migrated cells were determined by multi-detection microplate reader at excitation 458 nm and emission at 528 nm (BioTek Instrument, Inc., Winooski, VT, USA).

Biofilm formation was assessed as previously described [35 (link)]. Briefly, 24-well polystyrene cell culture plates (Costar 3524, Corning, NY, USA) were filled with KB broth and then inoculated with a 1:1000 dilution of each A. citrulli strain at a concentration of 3 × 10⁸ CFU/mL. The plates were incubated at 28 °C for 48 h without shaking. After incubation, 0.1% crystal violet was added to each well, and the plate was incubated for 30 min at room temperature. Each well was then gently washed three times with distilled water. To quantify biofilm formation, the stained biofilms were solubilized in 95% ethanol for 2 h, and then the OD₅₇₅ value of each stained cell suspension was measured with an Evolution 300 UV/VIS spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). There were three biological replicates for each A. citrulli strain in each experiment, and there were three independent replicates of this experiment.