Cell culture multi well plates

The PC12 cell line was obtained from Riken Cell Bank (Ibaraki, Japan). The cells were maintained in Dulbecco’s modified Eagle’s medium/F-12 supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Life Technologies, Franklin Lakes, NJ, USA) and 1% (v/v) penicillin–streptomycin. Cells were kept in an incubator at 37 °C in an atmosphere of 5% CO₂/95% air. For the ROT treatment, cells were seeded in cell culture multi-well plates (Thermo Scientific, Nunc, Naerum, Denmark) for 24 h and then treated with fresh medium containing NGF (final concentration of 50 ng/mL) for 48 h. Then, the cells were treated with ROT for 24 h to induce neurotoxicity. Cells were pretreated with ZPD for 8 h prior to exposure to ROT in the presence of NGF. The control group was treated with the same medium without ROT and ZPD.

Cells were grown in LB from a single colony isolated over LB plates to a mid-logarithmic phase (4 h at 37°C with shaking). For floating biofilm assays, the inoculum ratio was 1:1,000. Pellicle phenotypes in cultures grown at 23°C were observed. Photos of floating biofilms were acquired with a NikonD800 camera (Nikon, Tokyo, Japan) or a stereomicroscope (Zeiss, Oberkochenm, Germany) and images were optimized for contrast and brightness, with adjustments kept consistent. For evaluation of the effect of the surface area on pellicle formation, cells were re-diluted 1:1,000 in liquid MSgg medium in cell-culture multiwell plates (Thermo Scientific, Waltham, Massachusetts, USA) of 48, 24, 12, or 6 wells, containing a total volume of 0.85, 1.58, 2.95, or 8.6 ml of medium per-well, respectively, in order to reach comparable height without shaking. Plates were incubated at room temperature.

All chemicals used were of analytical grade. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was purchased from Aldrich (Steinheim, Germany). 2,6-dichloro-1,4-benzoquinone was purchased from Sigma Aldrich. HaCaT cells were used for the experiments. Dulbecco′s Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), L-Glutamine, penicillin, and streptomycin were purchased from Gibco, Thermo Fischer Scientific (Australia). Trypsin-EDTA 0.5% was purchased from Gibco, Thermo Fischer Scientific (America), and crystal violet was purchased from Merck (Darmstadt, Germany). Cell culture plates were purchased from Greiner Bio-One GmbH (Frickenhausen, Germany). Cell culture multiwell plates were purchased from Thermo Scientific (Denmark). Deuterated water (D₂O) and 3-(trimethylsilyl)-1-propionic-2, 2, 3, 3-d₄ acid sodium salt (TSP) were obtained from Deutero GmbH (Kastellaun, Germany). Double distilled water (DDW) was used to prepare all solutions. Phosphate Buffer Saline (PBS) was prepared with NaCl: 137.0 mM, KCl: 2.7 mM, Na₂HPO₄: 10.0 mM, KH₂PO₄: 1.8 mM. All solutions for the cell cultures were sterilized by autoclaving at 121^oC for 20 min, at 21 psi.