NSCs were dissociated and plated onto coverslips. The coverslips were prepared by first treating with 50 μg/mL poly-D-lysine (Sigma-Aldrich) and then coating with 5 μg/mL laminin (Life Technologies). NSCs were allowed to adhere for 24 h in growth medium before being changed to basal medium (growth medium without EGF, bFGF, or heparin) supplemented with 150 ng/mL of azIFN-γ, azIFN-γ without an azide-tag, or commercially available IFN-γ (Peprotech). After 8 d total, NSCs were fixed using methanol and stained using anti-βIII-tubulin (Covance, 1:500 dilution) with goat anti-mouse IgG Alexa-Fluor 546 (Life Technologies, 1:400 dilution) secondary antibody. The extent of neuronal differentiation was determined by counting the number of βIII-tubulin-positive cells and dividing by the total number of nuclei (stained by Hoechst 33342, Thermo Scientific). Imaging was performed on an Olympus IX81 with MetaMorph Advanced software (Molecular Devices).
Ham T.R., Farrag M, & Leipzig N.D. (2017). Covalent Growth Factor Tethering to Direct Neural Stem Cell Differentiation and Self-Organization. Acta biomaterialia, 53, 140-151.
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