CHO-K1 Cell Culture

Human Plasma Fibronectin (20 µg, Invitrogen) was labeled with 10 µg AlexaFluor 488 N-hydroxysuccinimide-ester (Invitrogen) at pH=8 and quenched with 0.31 µg

hydroxylamine (Sigma) in 1 M NaOH before mixing with and equal amount of unlabeled protein. Patterned monolayers were prepared on metal-coated glass coverslips (No. 1, 25 mm with 50 Å titanium metal and 100 Å gold) and 20 μg mL-1 (10 µg labeled, 10 µg unlabeled) fibronectin was absorbed onto the substrates at 37 °C for 1 h in Dulbecco’s Phosphate Buffered Saline (DPBS, Gibco/Life Technologies). Excess protein was removed by rinsing with DPBS (3x) and the coverslips were covered with fresh DPBS. CHO-K1 cells (ATCC) were detached using TrypLE Express (Invitrogen), followed by resuspension in Dulbecco’s Modified Eagle Medium (DMEM, low glucose 1X,

glutamax, 1 g L-1 D-glucose, 110 mg L-1 sodium pyruvate, 10% FBS, 1%

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Streptomycin Sulfate in 0.85% saline, Invitrogen), and counted using a hemacytometer (Bright-Line, Hausser Scientific). After rinsing the patterned coverslip with DPBS, approximately 30,000 or 90,000 cells were applied in 1 mL of DMEM. Plated cells were grown at 37 °C and 5% CO2. Cell and fluorescent protein images were visualized using

live-cell inverted microscopy with a Nikon TE2000-PFS microscope running NIS- Elements imaging software and equipped with an EXFO X-Cite UV illuminator, Photometrics CoolSNAP camera, and an In Vivo Scientific incubator chamber held at 37˚C, 5% CO2.

4.4.5. E18 Hippocampal Neuron Culture

CD-1 timed pregnancy mice (Charles River Laboratories) were sacrificed at day E18 and hippocampal pairs were microdissected from the embryonic mouse brains. E18 mouse hippocampi were stored in Hibernate E (BrainBits, LLC) for no longer than 72 hours and dissociated using a GentleMACS system in conjunction with the Neural Tissue

Dissociation Kit (P) and Dead Cell Removal Kit (Miltenyi Biotec). Mouse Laminin I (Trevigen) was labeled with AlexaFluor 488 as described above (Invitrogen) and deposited onto patterned substrates at 12 μg mL-1 (6 µg labeled, 6 µg unlabeled) for 1 h prior to plating. Following dissociation, neurons were counted using a hemacytometer (Bright-Line, Hausser Scientific) and seeded onto patterned substrates at a 45,000 cells mL-1 in Neurobasal medium supplemented with 2% B27 supplement, 1% Glutamax, 1% Penicillin/Streptomycin (Invitrogen). Heparin (sodium salt, porcine-derived heparin (Activity = 197.0 U mg-1, Calbiochem)) was dissolved in DPBS at a concentration of 20 mg mL-1, filter sterilized and diluted in fully-supplemented Neurobasal media (described

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above) to a final concentration of 2 µg mL-1. Cells were incubated at 37 °C, 5% CO2.

After two days in vitro, cultures were imaged by live-cell inverted microscopy. For the cell-neurite phenotype analysis, cells within the patterned region of the coverslip, with projections greater than 5 µm were counted and the path of their neurites evaluated.

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