Poly d lysine hydrobromide pdl

Fabrication of DopaFilm on glass substrates was initiated by a silane-based surface modification reaction. First, 35 mm gridded dishes (MatTek P35G-1.5–14 C-GRID) were cleaned sequentially with 200-proof ethanol and copious amounts of molecular biology grade water. The cleaned dishes were incubated with 1 mL of 1% (3-aminopropyl) triethoxysilane (APTES; Sigma Aldrich) in ethanol for 1 hr at room temperature. After silane functionalization, dishes were rinsed three times with 2.5 mL of molecular biology grade water. Subsequently, 500 µL of 15 ppm nanosensor solution was added to the glass coverslip of the dish dropwise, allowing the nanosensor to spread evenly. After an overnight incubation at room temperature, excess nanosensor solution was aspirated and carefully rinsed with 2.5 mL of molecular biology grade water. To facilitate neuronal growth on DopaFilm, 1 mL of 0.05 mg/mL poly-D-lysine hydrobromide (PDL; Sigma Aldrich P6407) was applied on top and incubated at room temperature for 1 hr. Finally, dishes were thoroughly rinsed three times with 2.5 mL of molecular biology grade water and stored in sterile 1 × PBS until seeding neurons. Neurons were seeded directly on the engineered surface immediately after removing storage PBS solution followed by washing with sterile water to remove excess salts.

The microfluidic chip consisted of one gel channel at the center and two medium channels on both sides. The trapezoidal-shaped micropillars were arrayed along the gel channel on both sides with a regular gap distance (200 µm) between them. The widths of the gel channel and medium channel were 1.3 and 1 mm, respectively. The total length of the channels was about 15 mm, and their depth was about 200 µm. The overall fabrication process of the 3D cell culture device followed a standard soft lithography protocol according to our previous reports 27 (link), 68 (link). After fabrication of Si master mold, the Sylgard 184 elastomer (Dow Corning, USA), poly-dimethylsiloxane (PDMS) with a curing agent was poured on the mold to a thickness of about 3 to 4 mm and baked at 80 °C for 2 h for polymerization. The PDMS replica was peeled off and the inlet and outlet holes (4 mm diameter) were punched out to connect to gel and medium channels. The PDMS layer was attached to the cover glass (24 × 24 mm) via plasma treatment. PDMS channels were coated with poly-D-lysine hydrobromide (PDL, 1 mg mL^-1, Sigma-Aldrich, USA) for 4 h to increase the electrostatic interactions between the gel and channel surfaces. The channels were rinsed and the microfluidic devices were dried in an oven at 80 °C.

For 2D cell migration experiments, cells were plated onto substrates coated with collagen I from rat tail (Sigma Aldrich, St. Louis), fibronectin from human plasma (Sigma Aldrich), or poly-d-lysine hydrobromide (PDL) (Sigma Aldrich). Collagen I and fibronectin were dissolved in PBS, and PDL was dissolved in sterile milliQ water. Twenty-four well glass bottom plates (MatTek, Ashland, MA) were coated with 300 µl (per well) of 20 μg/ml of the varying protein solutions and incubated for 1 h at 37°C. Following incubation, wells with collagen and fibronectin were washed 3 times with PBS, while wells with PDL were washed 3 times with sterile milliQ water. After washing the substrates, 1 × 10⁴ cells were plated into each well. Cells were imaged and analyzed as described below.

Unlabelled trimethylamine-N-oxide (TMAO), 1-methyl-4-phenylpyridinium (MPP⁺), and trimethoprim were obtained from Sigma-Aldrich (Taufkirchen, Germany). [³H]TMAO (60 Ci/mmol) and [³H]MPP⁺ (80 Ci/mmol) were obtained from Biotrend Chemikalien GmbH (Cologne, Germany). Poly-D-lysine hydrobromide (PDL) and sodium butyrate were purchased from Sigma-Aldrich (Taufkirchen, Germany). Cellstar 12-well cell culture plates and ThinCert^TM-TC Inserts (12 well, pore size 0.4 µm, translucent, filter area 1.1 cm²) were from Greiner Bio-One GmbH (Frickenhausen, Germany) or Sarstedt AG & Co. KG (Nümbrecht, Germany). Cell culture media supplements were obtained from Thermo Fisher Life Technologies GmbH (Darmstadt, Germany). All other chemicals and reagents, unless stated otherwise, were obtained from Carl Roth GmbH + Co. KG (Karlsruhe, Germany).