4.10. Neurite Outgrowth Assay

DRG neurons were cultured in OUB-diluted Sato medium (concentration 0, 25, 50, 75, and 100%) for 48 h and then immunostained with anti-TuJ1 antibody. The axonal length (the length of the longest neurite per TuJ1-positive neuron) and total neurite length per neuron were measured using a fluorescent microscope (eclipse 90i, Nikon) and a digital camera (DS-Ri1 and DS-U3, Nikon), as previously described [61]. Only neurites longer than 20 μm (about the diameter of a soma) and not in contact with other cells were measured. The axonal length and mean total neurite length were calculated from at least 30 neurons in each experiment.