Neurite Growth Assay in Mouse Neurons

SG at thoracic T1–T4 levels from 6- to 8-week-old mice were dissected, digested with collagenase I followed by Trypsin digestion. Dispersed neurons were cultured in complete neurobasal media (Fisher 10888022) with 1X B-27 (Fisher 17504044) and 200 µM l-glutamine (Fisher 25030149) in poly-d-lysine and Laminin coated dishes. Cells were cultured for 48 h with phosphate-buffered saline (PBS), NT-3 (100 ng/ml, R&D Systems, Minneapolis, MN) or CXCL10 (100 ng/ml, R&D Systems) treatment, re-plated, and further cultured for an additional 3–6 h with PBS, NT-3 (100 ng/ml) or CXCL10 (100 ng/ml) treatment. A similar NT-3 dose has been used in neuronal cultures previously^{78 (link)}. Cells were then immunostained with Alexa Fluor® 488-conjugated antibodies against neurite growth marker βIII-tubulin (1:400, AB15708A4, Millipore) (Supplementary Table 1)^{79 (link)}. Neurite growth was then quantitated by Image J (version 1.5.2) with Neuron J plugin (version 1.4.3)^{80 (link)}. Between 40 and 168 neurons were analyzed in each group, and neurite number and neurite length were normalized by the number of neurons in each group when doing comparisons between groups.