DRG Neuron Axon Outgrowth Assay

DRG cultures were prepared as described previously [9 (link)–11 (link)]. 8-10 week old C57BL/6J mouse DRGs were dissociated and cultured for 24 hours in 96-well PDL plates (BD BioCoat, Corning 354461) coated with laminin (Sigma L2020, 10 μg/ml in PBS, one hour at room temperature) or CSPG (Millipore, CC117). CSPG (3.3 ng/well in 50 μl PBS) was immobilized by drying overnight in a tissue culture hood and then rinsed with 50 μl of water to remove excess salt. DRG neurons were plated at a density of 2,500 neurons/cm² (833 neurons/well). Small molecules were added to the culture medium at the time of cell plating. After 24 hours of axon outgrowth, cultures were fixed and immunostained for βIII-tubulin (Sigma-Aldrich, T8660, 1:800) with an Alexa Fluor 488 secondary antibody (Invitrogen, A-11029, 1:500). Images were acquired using an ImageXpress^micro High Content Imaging System (Molecular Devices) with a 10X objective (four sites per well). Axon outgrowth was quantified using MetaXpress (Molecular Devices). The total axon length per neuron (axon length) is reported. n = 12-96 wells per condition.