Poly l lysine 0.01 solution

Biofilm development was performed in sterile polystyrene 24-well tissue culture plates (Greiner Bio-one, Frickenhausen, Germany).
In order to determine the effect of the oral products to be studied on bacterial viability, a multispecies biofilm formation was designed, where the substrates were incubated with poly-L-lysine solution 0.01% (Sigma-Aldrich, Darmstadt, Germany) for 1 h at 37 °C to promote and stabilize the biofilm.
The growth kinetics were evaluated by generating growth curves. The bacterial concentration was adjusted by spectrophotometry (optical density (OD) at 600 nm) in order to obtain a bacterial suspension containing 10⁵ CFU/mL (colony-forming units/mL) for S. mutans and 10⁶ CFU/mL each for F. nucleatum, P. gingivalis, and P. intermedia.
The culture medium used for the growth of S. mutans was brain–heart infusion broth (Scharlab, Barcelona, Spain) under CO₂ conditions at 37 °C for 24–48 h.
F. nucleatum, P. intermedia, and P. gingivalis were grown on fastidious anaerobe broth (EO labs, Bonnybridge, UK) under anaerobic conditions at 37 °C for 48–72 h for F. nucleatum and 72–96 h for P. intermedia and P. gingivalis.

Human RBC samples were prepared again in 3 different conditions. (1) Blank controls were RBCs in its original state without any incubation. (2) Negative samples were known D- RBCs incubated with 2% IgG anti-D at 37 °C for 30 minutes. (3) Positive samples were known D+ RBCs incubated with 2% IgG anti-D at 37 °C for 30 minutes. Glass Petri dishes were pre-treated with Poly-L-Lysine 0.01% solution, mol wt 70,000–150,000 (Sigma-Aldrich, USA) in accordance with the Poly-L-Lysine Cell Attachment Protocol provided by the manufacturer (George Sitterley, Biofiles 2008, 3.8, 12.). The different RBC samples are left to attach on the Poly-L-Lysine treated glass petri dish submerged in phosphate buffered saline (Sigma-Aldrich, USA) for 10 minutes and washed gently with PBS to remove loose unattached cells. RBCs attached to the glass Petri dish were then resubmerged in Celpresol (CSL, Australia).
Force mapping studies were conducted using a JPK Nanowizard 3 AFM with the contact mode force mapping option. Scan areas of 1 μm × 1 μm and 0.1 μm × 0.1 μm on random areas of the RBC surface were conducted to collect interaction force data between the anti-IgG on the cantilever and the IgG antibody, if present on cell surface. More than 3 cells for each sample was scanned and each cell were scanned for more than 3 times. Each scan was performed with a resolution of 16 × 16 pixels.

Organoids were removed from the Matrigel^® (Corning Inc., Corning, NY, USA, CACB356231) domes and collected in ice-cold DMEM/F12 (Invitrogen, 12634010) and pelleted through centrifugation at 500× g for 5 min. Pellets were then re-suspended in growth factor conditioned medium and plated onto Poly-L-Lysine (0.01% solution, Sigma-Aldrich, P4707) coated plates (Corning Inc., 3916). Organoids were collected and suspended at a density of approximately 1000 organoids per 1 mL of media. This suspension was used to seed wells in a 96-well plate such that after opening, 50% of the surface area is covered (Figure S1). Media were changed one-day post-seeding. All functional studies were conducted two days after organoid plating. With the apical application of specific electrochemical gradients, ligands and inhibitors, we selectively measure the apical electrogenic membrane protein function.

Atomic force microscope (Asylum
MFP-3D, Oxford instruments), standard air-tapping mode (silicon probes
Ted Pella, silicon probes), 300 kHZ resonance frequency, and 40 N/m
force, was used. We used mica surface (Ted Pella, freshly cleaved)
layered by 20 μL of poly-l-lysine 0.01% solution (Sigma-Aldrich),
and, upon wash with water, 20 μL of GO suspension (100 μg/mL)
was drop casted. Gwyddion software (http://gwyddion.net, version 2.56) was used to image processing.
ImageJ software (https://imagej.nih.gov) with AFM images 5 μm × 5 μm height distribution
analysis allowed was used to obtain the lateral dimensions values.

HeLa (RRID: CVCL_0030) and HEK293 (RRID: CVCL_0045) cells were obtained from Leibniz Forschungsinstitut DSMZ and regularly tested for mycoplasma contamination. LRRC8^-/- HEK293 (HEK293 KO) cells deficient in all five LRRC8 subunits (Lutter et al., 2017 (link)) were kindly provided by T.J. Jentsch. Cells were grown in DMEM (Pan-Biotech) supplemented with 10% fetal calf serum at 37°C in 5% CO₂. For imaging experiments without simultaneous electrophysiology, cells were plated in 35 mm glass bottom dishes (MatTek), coated with poly-L-lysine 0.01% solution (Sigma-Aldrich) for HEK293 cells. For electrophysiology, cells were plated on poly-L-lysine-coated 25 mm coverslips. Cells were transfected with FuGENE 6 (Promega) according to the supplier’s manual. For co-expression, constructs were co-transfected at equimolar ratios.