Cm5 biacore chip

Nanoparticle binding affinities to Fn14 extracellular domain was evaluated by SPR using a Biacore 3000 instrument at 25°C. The Fn14 extracellular domain (Cell Sciences, Canton, MA) was conjugated to a CM5 Biacore chip, with three different Fn14 ligand RU values ranging from 50 to 300. The first flow path (Fc1) was activated and blocked with ethanolamine to serve as a reference for each binding run, as suggested per manufacturer’s protocol. The running buffer was degassed 10 mM HEPES buffer (pH 7.4) containing 150 mM NaCl, 0.05% surfactant P-20 with 50 µM EDTA (HBS-P+). For SPR experiments, samples were run at a flow rate of 20 µL/min with an injection time of 3 min followed by a 2.5 min wait time for dissociation, before chip regeneration with either 100 mM phosphoric acid, pH 3 or 10 mM glycine, pH 1.75 (GE Healthcare). IgG isotype (25 nM) was used as a negative control and ITEM4 (25 nM) as a positive control. Nanoparticle binding was assayed with particle concentrations ranging between 1 µg/mL and 200 µg/mL diluted in running buffer. Data were analyzed using Biacore 3000 Evaluation Software, where data from Fc1 was subtracted from the Fc2, Fc3, and Fc4 data to give the final sensorgrams. Equilibrium binding affinities (K_D) were calculated as previously described [38 (link)].

Nanoparticle binding affinities to Matrigel were evaluated by SPR using a Biacore 3000 instrument at 25 °C. Matrigel was diluted to 100 μg/mL in acetate buffer pH 4.0 and was conjugated to a CM5 Biacore chip with RU value of ~2000. The first flow path (Fc1) was activated and blocked with ethanolamine to serve as a reference for each binding run, as suggested per manufacturer’s protocol. The running buffer, 10 mM HEPES buffer (pH 7.4) containing 150 nM NaCl, 0.05% surfactant Tween 20 with 50 μM EDTA (HBS-P+), was degassed prior to use. For SPR experiments, samples were run at a flow rate of 20 μL/min with an injection time of 3 min followed by a 2 min wait time for dissociation, before chip regeneration with 10 mM glycine, pH 1.75 (GE Healthcare). Nanoparticle binding was assayed with particle concentrations of 1 mg/mL diluted in running buffer. Data were analyzed using Biacore 300 Evaluation Software, where data from Fc1 was subtracted from the Fc2, Fc3, and Fc4 data to give the final sensorgrams.

Brain extracellular matrix (ECM) proteins were isolated from freshly collected mouse brain as previously described [39 (link)]. Briefly, resected whole mouse brain was frozen for at least 24 h at −80°C and subsequently thawed and decellularized in a series of steps: ultrapure water (16 h at 4°C), 0.02% trypsin/0.05% EDTA (1 h at 37°C), 3% Triton-X 100 (1 h), 1.0 M sucrose (15 min), ultrapure water (15 min), 4% deoxycholate (1 h), 0.1% periacetic acid in 4% ethanol (2 h), 1X PBS (15 min), ultrapure water (15 min), and 1X PBS (15 min). The decellularized proteins were filtered (0.2 µm filter) to remove insoluble proteins and then frozen and stored at −80°C until use.
The isolated mouse brain ECM proteins were conjugated to the second flow channel (Fc2) of a CM5 Biacore chip with ligand RU values ranging from 140 to 250. The first flow path was activated and blocked with ethanolamine to serve as a reference for each binding run. For binding experiments, samples were assayed at a flow rate of 20 µL/min with an injection time of 3 min followed by a 2.5 min wait time for dissociation, before chip regeneration with either 100 mM phosphoric acid, pH 3 or 10 mM glycine, pH 1.75 (GE Healthcare). Nanoparticle binding was assayed with particle concentrations ranging between 1 µg/mL and 200 µg/mL, diluted in running buffer.